Acylsulfonamides and Processes for Producing the Same

ABSTRACT

The present disclosure relates to acylsulfonamides and processes for their preparation. The processes involve a target-guided synthesis approach, whereby a thioacid and a sulfonyl azide are reacted in the presence of a biological target protein, a Bcl-2 family protein, to form the acylsulfonamide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.Nos. 61/030,753 and 61/030,756, filed Feb. 22, 2008, which are herebyincorporated by reference in their entirety, including any figures,tables, and drawings.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States government support underGrant No. 07KN-08 awarded by the James and Esther King BiomedicalResearch Program and Grant No. P01CA118210 awarded by the NationalCancer Institute, National Institutes of Health. The United Statesgovernment has certain rights in the invention.

BACKGROUND

The present disclosure generally relates to acylsulfonamides andprocesses for their preparation. The disclosure also relates to akinetically controlled target-guided synthesis approach for thediscovery and development of small molecules.

Combinatorial chemistry and parallel synthesis are the tools commonlyutilized for lead compound identification and optimization. However,even though in the last two decades combinatorial chemistry and parallelsynthesis have gone hand in hand with the dramatic advances oftechnology for rapid production, handling and screening of large numbersof compounds, they are often accompanied by challenges such as theefficiency of library synthesis, the purity of each library member, andthe unambiguous identification of lead compounds in the screening ofeach library member against a particular biological target. In the lastdecade, fragment-based lead compound discovery or target-guidedsynthesis (TGS) approaches have been developed in which the biologicaltarget is actively engaged in the design and the synthesis of its ownenzyme inhibitory compounds. To date, target-guided synthesis hasexclusively been applied for enzymatic targets only. See, e.g., Manetschet al., Journal of the American Chemical Society 2004, 126, 12809-12818;Sharpless et al., Expert Opin. Drug Discovery 2006, 1, 525-538; and Kolbet al., U.S. Patent Publication No. 2006/0269942.

Among a variety of proteins, the Bcl-2 family of proteins, whichconsists of both anti- and pro-apoptotic molecules, in particular, canplay an important role in the regulation of the intrinsic(mitochondrial) pathway of apoptosis. The anti-apoptotic Bcl-2 familyproteins (e.g., Bcl-2, Bcl-X_(L), Mcl-1) inhibit the release of certainpro-apoptotic factors from mitochondria, whereas pro-apoptotic Bcl-2family members, which can be further separated into two subgroups, themultidomain BH1-3 proteins (Bax and Bak) and the BH3-only proteins(e.g., Bad, Bim, and Noxa), induce the release of mitochondrialapoptogenic molecules into the cytosol. Although the precise biochemicalmechanisms by which Bcl-2 family proteins exert their influence on celllife and death remains far from clear, the relative ratios of pro- andanti-apoptotic Bcl-2 family proteins determine the ultimate sensitivityor resistance of cells to a wide variety of apoptotic signals.

Evidence has accumulated that the majority of human cancers overexpressthe pro-survival Bcl-2 family proteins, which not only contribute tocancer progression by preventing normal cell turnover, but also rendercancer cells resistant to current cancer treatments. For example, highlevels of Bcl-2 are found in ˜30% to 60% of prostate cancer, ˜60% to 90%of breast cancer, ˜20% to 40% of non-small cell lung cancer, ˜60% to 80%of small cell lung cancer, ˜50% to 100% of colorectal cancer, ˜65% ofmelanoma, ˜30% of neuroblastomas, and ˜80% of B cell lymphomas.Similarly, Bcl-X_(L) is overexpressed in ˜100% of hormone-refractoryprostate cancer, ˜40% to 60% of breast cancer, ˜80% of colorectalcancer, ˜90% of melanoma, ˜90% of pancreatic cancer, and ˜80% ofhepatocellular carcinoma. It has been shown that overexpression of Bcl-2and/or Bcl-X_(L) renders cancer cells resistant to most of the currentlyavailable chemotherapeutic drugs as well as radiation therapy.Therefore, it is an attractive strategy to design and develop a newclass of anticancer drugs that specifically target the anti- andpro-apoptotic functions of the Bcl-2 family proteins.

SUMMARY OF THE DISCLOSURE

Among the various aspects of the present disclosure is the provision ofa target-guided synthesis approach for the discovery and development ofsmall molecules, and in particular acylsulfonamides.

Briefly, therefore, the present disclosure is directed to a process forthe preparation of an acylsulfonamide (3), the process comprisingreacting a thioacid (1) with a sulfonyl azide (2) in the presence of aprotein of the Bcl-2 family, wherein the thioacid (1), the sulfonylazide (2), and the acylsulfonamide (3) correspond to Formulae (1), (2),and (3):

Z₁ is hydrocarbyl, substituted hydrocarbyl, heteroaryl, or heterocyclo;and

Z₂ is hydrocarbyl, substituted hydrocarbyl, heteroaryl, or heterocyclo.

Another aspect of the disclosure is directed to an acylsulfonamide (3)having the formula:

wherein

Z₁ has the formula:

Z₂ has the formula:

Z₁₁ and Z₁₃ are alkyl, substituted alkyl, —OH, —OR_(Z), —COOH,—COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z),—SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo, among others, whereineach occurrence of R_(Z) is substituted or unsubstituted alkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedaralkyl;

Z₂₂ is —N(Z₂₂₀)(Z₂₂₁) or —CH₂—N(Z₂₂₀)(Z₂₂₁), wherein Z₂₂₀ and Z₂₂₁ areindependently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, or Z₂₂₀ and Z₂₂₁ together with the nitrogen atom to which they areattached, form a substituted or unsubstituted alicyclic, bicyclic, aryl,or heterocyclic moiety; and

Z₁₀, Z₁₂, Z₁₄, Z₂₀, Z₂₁, Z₂₃, and Z₂₄ are hydrogen.

Other aspects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the ribbon structure of a Bcl-X_(L)-Bak complex andthe surface representation of the binding pocket of Bcl-X_(L) bound tothe Bak peptide.

FIG. 2 illustrates exemplary steps of conventional lead discovery andtarget-guided synthesis protocols.

FIG. 3 illustrates the binding pockets of the Bcl-X_(L)-Bak complex.

FIG. 4 is an LC/MS trace illustrating the comparison between incubationsof (SZ4 and TA2) measured by LC/MS-SIM Mode and LC/MS-Scan Mode. A)Incubation of (SZ4) and (TA2) without Bcl-X_(L) measured by LC/MS-SIMmode; B) Incubation of (SZ4) and (TA2) with 2 μM Bcl-X_(L) measured byLC/MS-SIM mode; C) Incubation of (SZ4) and (TA2) with 2 μM Bcl-X_(L)measured by LC/MS-Scan mode.

FIG. 5 is an LC/MS trace illustrating the incubations of (SZ1) and (TA2)and incubations of (SZ2) and (TA2). A) Incubation of (SZ1) and (TA2)without Bcl-X_(L); B) Incubation of (SZ1) and (TA2) with 2 μM Bcl-X_(L);C) Incubation of (SZ5) and (TA2) without Bcl-X_(L); D) Incubation of(SZ5) and (TA2) with 2 μM Bcl-X_(L).

FIG. 6 is an LC/MS trace illustrating the incubations of (SZ4) and (TA2)with bovine erythrocyte carbonic anhydrase II, concanavalin A and mAChE.A) Incubation of (SZ4) and (TA2) without proteins; B) Incubation of(SZ4) and (TA2) with 2 μM of bCAII; C) Incubation of (SZ4) and (TA2)with 2 μM of ConA. D) Incubation of (SZ4) and (TA2) with 2 μM of mAChE.E) Incubation of (SZ4) and (TA2) with 2 μM of Bcl-X_(L).

FIG. 7 is an LC/MS trace illustrating the incubations of (SZ4) and (TA2)with Bak BH3 peptide for 24 hours. A) Incubation of (SZ4) and (TA2)without Bcl-X_(L) or Bak BH3 peptide; B) Incubation of (SZ4) and (TA2)with 20 μM Bak BH3 peptide and without no Bcl-X_(L).

FIG. 8 is an LC/MS trace illustrating Bcl-X_(L)-templated incubationscontaining Bim, mutant Bim and mutant Bak. A) Incubation of (SZ4) and(TA2) without Bcl-X_(L); B) Incubation of (SZ4) and (TA2) with 2 μMBcl-X_(L); C) Incubation of (SZ4) and (TA2) with 2 μM Bcl-X_(L) and 20μM Bak BH3 peptide; D) Incubation of (SZ4) and (TA2) with 2 μM Bcl-X_(L)and 20 μM of mutant Bak; E) Incubation of (SZ4) and (TA2) with 2 μMBcl-X_(L) and 20 μM of Bim; F) Incubation of (SZ4) and (TA2) with 2 μMBcl-X_(L) and 20 μM of mutant Bim.

FIG. 9 is an LC/MS trace illustrating incubations of (SZ4) and (TA2)with Bim, mutant Bim and mutant Bak (no Bcl-X_(L)). A) Incubation of(SZ4) and (TA2) without peptides; B) Incubation of (SZ4) and (TA2) with20 μM Bim; C) Incubation of (SZ4) and (TA2) with 20 μM of mutant Bim; D)Incubation of (SZ4) and (TA2) with 20 μM of mutant Bak.

FIG. 10 is an LC/MS trace illustrating the suppression ofBcl-X_(L)-templated incubations with Bak BH3 Peptide. Incubation sampleswere kept for six hours at 37° C. A) Incubation of (SZ4) and (TA2)without Bcl-X_(L); B) Incubation of (SZ4) and (TA2) with 2 μM Bcl-X_(L);C) Incubation of (SZ4) and (TA2) with 2 μM Bcl-X_(L) and 20 μM Bak BH3peptide; D) Incubation of (SZ4) and (TA2) with 2 μM Bcl-X_(L) and 10 μMBak BH3 peptide; E) Incubation of (SZ4) and (TA2) with 2 μM Bcl-X_(L)and 5 μM Bak BH3 peptide; F) Incubation of (SZ4) and (TA2) with 2 μMBcl-X_(L) and 2 μM Bak BH3 peptide.

FIG. 11 is an LC/MS trace illustrating Bcl-X_(L) incubations containingsulfonylazides (SZ1)-(SZ6) and thioacid (TA2). A) Incubation of(SZ1)-(SZ6) and (TA2) without Bcl-X_(L); B) Incubation of (SZ1)-(SZ6)and (TA2) with 404 Bcl-X_(L); C) Synthesized compound (SZ4TA2) asreference.

FIGS. 12-19 are LC/MS trace illustrating other sulfonyl azide andthioacid combinations: (SZ7) and (TA2) (FIG. 12); (SZ9) and (TA5) (FIG.13); (SZ10) and (TA2) (FIG. 14); (SZ15) and (TA3) (FIG. 15); (SZ15) and(TA8) (FIG. 16); (SZ16) and (TA4) (FIG. 17); (SZ16) and (TA8) (FIG. 18);and (SZ17) and (TA7).

DETAILED DESCRIPTION

Among other things, the present disclosure relates to a fragment-basedlead compound discovery method, in which the biological target, e.g., amember of the Bcl-2 family of proteins, is directly involved in theassembly of its own bidentate ligand from two or more smaller reactivefragments or scaffolds. The methods described herein are versatiletarget-guided synthesis approaches for probing adaptive regions on/inbiological targets, and in particular Bcl-2 family protein targets, andcan be exploited as an innovative means to identify and optimize smallmolecules interacting with such biological targets. The target-guidedsynthesis methods are successful, in part, due to: (a) the nature of thechemical reaction combining the two fragments or scaffold compounds intoa single molecule; and (b) the use of reactive fragments showing low tohigh affinity towards binding pockets or surfaces of the biologicaltargets.

Another key component of the processes described herein is thereactivity of the utilized reactions; specifically, the functionalitieson the building block or scaffold compounds can be tuned not only to theparticular biological target, but also to speed up or slow downreactivity with the biological target, improving the formation ofbidentate ligand(s) displaying good affinity to the biological target.Among other things, the processes described herein address certainlimitations of the target-guided synthesis methods reported thus far;compared to the reported target-guided synthesis methods for thescreening of enzymes, the discovery of protein interactions is morechallenging because biological target/interfaces have relatively shallowbinding sites on their surfaces, thus permitting only weak bindingaffinity for reactive fragments. This often translates to shortresidence times for these fragments within the binding cavities. Forthese and other reasons, previously reported target-guided synthesismethods poorly succeed or even fail in discovery attempts.

As noted above, the processes described herein utilize certainstructural moieties or scaffolds having activity against Bcl-2 familyprotein interactions (also referred to as protein-protein interactionmodulation (PPIM)). PPIM activity can be achieved as described herein bycompound design including one or two of the aforementioned structuralmoieties in the same compound. Each scaffold portion is designed to bindto one or more subpockets of a biological target, e.g., a Bcl-2 familyprotein. In a particular embodiment, the compounds prepared by thetarget-guided synthesis methods described herein are acylsulfonamidecompounds that are capable of binding to one or more of the subpocketsof a Bcl-2 family (e.g., Bcl-X_(L), the binding subpockets of which aredesignated as P1, P2, P3, P4, and P5) (see, e.g., FIG. 3)). In aparticular embodiment, the acylsulfonamide compounds target the P4and/or P5 region of Bcl-X_(L).

Compared to the previously reported target-guided synthesis screeningmethods for enzyme inhibitors, the target-guided synthesis approachesdescribed herein utilize reactions with superior reactivity profiles,enabling the use of traditionally weak affinity small molecules asrelatively reactive fragments for the discovery and optimization ofligands and compounds. The enhanced reactivity is due, in part, to theuse of more reactive functionalities for the chemical reaction(s) thatcombines the two fragments into a larger molecule.

Among other things, the present disclosure relates to the preparation ofacylsulfonamides. According to the processes described herein, at leastone (and typically two or more) thioacid is incubated or reacted with atleast one (and typically two or more) sulfonyl azide in the presence ofa protein of the Bcl-2 family to form an acylsulfonamide. In certainembodiments, the protein is Bcl-X_(L). In certain other embodiments, theprotein is Mcl-1. In general, the reaction involves an amidationreaction between electron-poor thioacids and sulfonyl azides or betweenthioacids and electron-rich sulfonyl azides. See, e.g., Shangguan et al.J. Am. Chem. Soc. 2003, 125, 7754-7755.

The acylsulfonamide-forming reaction described herein is generallyillustrated in Reaction Scheme (1), wherein Z₁ and Z₂ are described inconnection with Formulae (1), (2), and (3) below:

As shown, the thioacid (1) is reacted with a sulfonyl azide (2) in thepresence of a Bcl-2 family protein. Usually, the reaction involves apool or library of two or more thioacids (1), and a corresponding poolor library of two or more sulfonyl azides (2). The reaction is typicallycarried out at relatively ambient or slightly higher temperatures, whichenhances the rate of the ligation reaction. The acylsulfonamide-formingreaction is typically carried out at a temperature of at least 20° C.,preferably at least 25° C., and more preferably 30-40° C. Reaction timescan range from about 1 hour to several days; e.g., from about 1 hour toabout 48 hours (e.g., 6-12 hours, 12-36 hours, or 24-72 hours).

The reaction mixture for preparing the acylsulfonamide (3) according tothe methods described herein typically comprises the thioacid (1) (or alibrary thereof), the sulfonyl azide (2) (or a library thereof), thebiological target, and an aqueous buffer medium, which may be optimizeddepending on the particular thioacid(s) (1), sulfonyl azide(s) (2), andBcl-2 family protein selected for the reaction. Preferably, the bufferis an aqueous physiological buffer that is compatible with biologicalmaterials. Buffers useful in the preparation of acylsulfonamidesaccording to the processes described herein include but are not limitedto phosphate-, citrate-, sulfosalicylate-, and acetate-based buffers, orother organic acid-based buffers. Still other buffers include ADAbuffer, ACES buffer, BES buffer, BIS TRIS buffer, DIPSO buffer, HEPESbuffer, MOPS buffer, MOPSO buffer, PIPES buffer, TES buffer, Trisbuffer, Tricine buffer, TRISMA buffer, and the like. A more completelist can be found in the United States Pharmacopeia. In one embodiment,the buffer is a phosphate buffer (e.g., sodium phosphate, potassiumphosphate). In certain preferred embodiments the buffering agent will bepresent in an amount sufficient to provide a pH ranging from about 6.0to 9.5, more preferably pH 7.4. Other agents that may be present in thebuffer medium include chelating agents, such as EDTA, EGTA, and thelike.

Thioacids

In accordance with the present methods, a thioacid (or a library ofthioacids) is reacted with a sulfonyl azide (or a library of sulfonylazides) in the presence of a biological target molecule; in preferredembodiments, the biological target molecule is a Bcl-2 family protein.In general, the Bcl-2 family protein acts as a template for theformation of the acylsulfonamide. As noted above in connection withReaction Scheme (1), the thioacid corresponds to Formula (1):

wherein

Z₁ is hydrocarbyl, substituted hydrocarbyl, heteroaryl, or heterocyclo.

Typically, such hydrocarbyl substituents for Z₁ contain from 1 to 20carbon atoms and may be linear, branched, or cyclic, and saidsubstituted hydrocarbyl, heteroaryl, and heterocyclo moieties for Z₁ maybe substituted with one or more of ═O, —OH, —OR_(Z), —COOH, —COOR_(Z),—CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z), —SO₂R_(Z),—SO₂H, —SOR_(Z), heterocyclo, and halo (including F, Cl, Br and I),among others, wherein each occurrence of R_(Z) may be hydrocarbyl orsubstituted hydrocarbyl (e.g., substituted or unsubstituted alkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedaralkyl).

Although Z₁ is hydrocarbyl, substituted hydrocarbyl, heteroaryl, orheterocyclo, in certain embodiments Z₁ is aryl, substituted aryl, orheteroaryl. In the embodiments in which Z₁ is aryl or substituted aryl,for example, Z₁ may have the formula:

wherein Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄ are independently hydrogen,hydroxyl, protected hydroxyl, halo, hydrocarbyl, substitutedhydrocarbyl, heterocyclo, heteroaryl, alkoxy, alkenoxy, alkynoxy,aryloxy, arylalkoxy (heterocyclo)alkoxy, trihaloalkoxy, amino, amido, orcyano, or two of Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄, together with the carbonatoms to which they are attached, form a fused carbocyclic (e.g.,napthyl) or heterocyclic ring. In one embodiment, Z₁ corresponds to thearyl or substituted aryl structure illustrated above and Z₁₀, Z₁₁, Z₁₂,Z₁₃, and Z₁₄ are independently hydrogen, amino, alkoxy, nitro, ortrihalomethoxy (e.g., trifluoromethoxy); more preferably in thisembodiment, Z₁₀ and Z₁₄ are hydrogen and Z₁₁, Z₁₂, and Z₁₃ areindependently hydrogen, amino, alkoxy, nitro, or trihalomethoxy. In oneparticular embodiment, Z₁ is a substituted phenyl or napthyl moiety,with substituents in the ortho-, para-, or meta-positions; morepreferably in this embodiment, Z₁ is a para-substituted phenyl ornapthyl moiety; thus, for example, at least Z₁₁ and Z₁₃ in the abovestructure are substituted with alkyl, substituted alkyl, —OH, —OR_(Z),—COOH, —COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH,—SR_(Z), —SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo (including F,Cl, Br and I), among others, wherein each occurrence of R_(Z) may behydrocarbyl or substituted hydrocarbyl (e.g., substituted orunsubstituted alkyl, substituted or unsubstituted aryl, or substitutedor unsubstituted aralkyl). Typically in this embodiment, Z₁₀, Z₁₂, andZ₁₄ are hydrogen.

In the embodiments in which Z₁ corresponds to the aryl or substitutedaryl structure illustrated above and where one or more of Z₁₀, Z₁₁, Z₁₂,Z₁₃, and Z₁₄ are hydrocarbyl, for example, they may be independentlyalkyl, alkenyl, alkynyl, aryl, alkaryl, or aralkyl. Typically, suchsubstituents contain from 1 to 20 carbon atoms and may be linear,branched, or cyclic. By way of example, the Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄substituents may be selected from methyl, ethyl, n-propyl, cyclopropyl,isopropyl, n-butyl, cyclobutyl, isobutyl, s-butyl, n-pentyl, isopentyl,cyclopentyl, n-hexyl, isohexyl, cyclohexyl, benzyl, phenyl, and napthyl.Where one or more of Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄ are substitutedhydrocarbyl, for example, they may be independently substituted alkyl,substituted alkenyl, substituted alkynyl, substituted aryl, substitutedalkaryl, or substituted aralkyl. Similar to the hydrocarbyl moieties,these substituents may contain 1 to 20 carbon atoms and may be linear,branched, or cyclic; one or more hydrogen atoms of the substitutedhydrocarbyl moieties, however, are replaced with a different substituentsuch as, for example, ═O, —OH, —OR_(Z), —COOH, —COOR_(Z), —CONH₂, —NH₂,—NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z), —SO₂R_(Z), —SO₂H, —SOR_(Z),heterocyclo, and halo (including F, Cl, Br and I), among others, whereineach occurrence of R_(Z) may be hydrocarbyl or substituted hydrocarbyl(e.g., substituted or unsubstituted alkyl, substituted or unsubstitutedaryl, or substituted or unsubstituted aralkyl).

Where Z₁ corresponds to the aryl or substituted aryl structureillustrated above and where one or more of Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄are amino, for example, the amino moiety may have the formula:—N(Z_(X))(Z_(Y)) wherein Z_(X) and Z_(Y) are independently hydrogen,hydrocarbyl, substituted hydrocarbyl, heteroaryl, heterocyclo, or anamino protecting group, or Z_(X) and Z_(Y), together with the nitrogenatom to which they are attached, form a substituted or unsubstitutedalicyclic, bicyclic, aryl, heteroaryl, or heterocyclic moiety, typicallyhaving 3 to 10 atoms in the ring.

In one particular embodiment, Z₁ has the formula:

wherein A is phenyl or a five- or six-membered aromatic carbocyclic orheterocyclic ring wherein from one to three carbon atoms may be replacedby a heteroatom selected from N, O, or S, and wherein A is substitutedwith Z₁₀₀ and Z₁₀₁ through ring carbon atoms or ring heteroatoms, andZ₁₀₀ and Z₁₀₁ are independently hydrogen, hydrocarbyl, substitutedhydrocarbyl, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy,heterocyclo(alkoxy), or halo. Where Z₁₀₀ and Z₁₀₁ are hydrocarbyl orsubstituted hydrocarbyl, for example, they may be substituted orunsubstituted (straight, branched, or cyclic) alkyl, alkenyl, alkynyl,aryl, aralkyl, or arylalkenyl, wherein the substituents for such groupsmay be, for example, ═O, —OH, —OR_(Z), —COOH, —COOR_(Z), —CONH₂, —NH₂,—NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z), —SO₂R_(Z), —SO₂H, —SOR_(Z),heterocyclo, and halo (including F, Cl, Br and I), among others, whereineach occurrence of R_(Z) may be hydrocarbyl or substituted hydrocarbyl(e.g., substituted or unsubstituted alkyl, substituted or unsubstitutedaryl, or substituted or unsubstituted aralkyl). In one particularembodiment, Z₁₀₀ and Z₁₀₁ are selected from hydrogen, alkyl, aryl,arylalkenyl, arylalkoxy, cycloalkenyl, cycloalkyl, halo, heterocyclo, or(heterocyclo)alkoxy. Where Z₁₀₀ and/or Z₁₀₁ are heterocyclo, forexample, they may be selected from substituted or unsubstitutedmorpholino, pyran, tetrahydropyran, piperazinyl, piperidinyl,tetrahydropyridinyl, pyrrolidinyl, 1,4-diazepanyl, and azepinyl.

In another particular embodiment, Z₁ has the structure:

wherein A is a five-, six-, or seven-membered non-aromatic ringcontaining a nitrogen atom wherein from zero to two carbon atoms arereplaced by a heteroatom selected from N, O, or S, and wherein A issubstituted with Z₁₀₀ and Z₁₀₁ through ring carbon atoms or ringheteroatoms, and Z₁₀₀ and Z₁₀₁ are independently hydrogen, hydrocarbyl,substituted hydrocarbyl, heterocyclo, alkoxy, alkenoxy, alkynoxy,aryloxy, heterocyclo(alkoxy), or halo. In accordance with theseembodiments, for example, Z₁ may be a substituted or unsubstitutedpiperazine, piperidine, tetrahydropyridine, pyrrolidine, pyrroline,1,4-diazepane, or azepane moiety. Where Z₁₀₀ and Z₁₀₁ are hydrocarbyl orsubstituted hydrocarbyl, for example, they may be substituted orunsubstituted (straight, branched, or cyclic) alkyl, alkenyl, alkynyl,aryl, aralkyl, or arylalkenyl, wherein the substituents for such groupsmay be, for example, ═O, —OH, —OR_(Z), —COOH, —COOR_(Z), —CONH₂, —NH₂,—NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z), —SO₂R_(Z), —SO₂H, —SOR_(Z),heterocyclo, and halo (including F, Cl, Br and I), among others, whereineach occurrence of R_(Z) may be hydrocarbyl or substituted hydrocarbyl(e.g., substituted or unsubstituted alkyl, substituted or unsubstitutedaryl, or substituted or unsubstituted aralkyl). In one particularembodiment, Z₁₀₀ and Z₁₀₁ are selected from hydrogen, alkyl, aryl,arylalkenyl, arylalkoxy, cycloalkenyl, cycloalkyl, halo, heterocyclo, or(heterocyclo)alkoxy. Where Z₁₀₀ and/or Z₁₀₁ are heterocyclo, forexample, they may be selected from substituted or unsubstitutedmorpholino, pyran, tetrahydropyran, piperazinyl, piperidinyl,tetrahydropyridinyl, pyrrolidinyl, 1,4-diazepanyl, and azepinyl.

As noted above, in certain embodiments, Z₁ is heteroaryl. According tothese embodiments, for example, Z₁ may be substituted or unsubstitutedfuryl, thienyl, pyridyl, oxazolyl, isoxazolyl, imidazolyl, pyridyl,pyrimidyl, purinyl, triazolyl, or thiazolyl. In one particularembodiment, Z₁ is phenyl, substituted phenyl, substituted alkyl, orsubstituted or unsubstituted furyl, thienyl, pyridyl, pyridinyl,oxazolyl, imidazolyl, pyridyl, pyrimidyl, purinyl, triazolyl, orthiazolyl; more preferably in this embodiment, Z₁ is phenyl, substitutedphenyl, pyridinyl, substituted pyridinyl, furyl, or substituted furyl.In these embodiments, the substituents for the substituted groups maycorrespond to those described above in connection with Z₁₀₀ and Z₁₀₁.

In another embodiment, Z₁ is heterocyclo. In accordance with thisembodiment, for example, Z₁ may be substituted or unsubstitutedmorpholino, pyran, tetrahydropyran, piperazinyl, piperidinyl,tetrahydropyridinyl, pyrrolidinyl, pyrrolinyl, 1,4-diazepanyl, orazepinyl. In these embodiments, the substituents for the substitutedgroups may correspond to those described above in connection with Z₁₀₀and Z₁₀₁.

In another embodiment, Z₁ is alkyl or substituted alkyl. In accordancewith this embodiment, therefore, Z₁ may be —(CH₂)_(x)—Z₁₀₂ wherein Z₁₀₂is hydrogen, hydroxyl, protected hydroxyl, heterocyclo, amino, amido,alkoxy, aryloxy, cyano, nitro, thiol, or an acetal, ketal, ester, ether,or thioether, and x is 1, 2, or 3. Where Z₁₀₂ is amino, for example,Z₁₀₂ may have the formula: —N(Z_(X))(Z_(Y)) wherein Z_(X) and Z_(Y) areindependently hydrogen, hydrocarbyl, substituted hydrocarbyl,heteroaryl, heterocyclo, or an amino protecting group, or Z_(X) andZ_(Y), together with the nitrogen atom to which they are attached, forma substituted or unsubstituted alicyclic, bicyclic, aryl, heteroaryl, orheterocyclic moiety, typically having 3 to 10 atoms in the ring. In oneembodiment in which Z₁₀₂ is amino, for example, Z₁₀₂ is a substituted orunsubstituted piperidine, piperazine, or tetrahydroisoquinoline;according to certain embodiments in which Z₁₀₂ is atetrahydroisoquinoline, the tetrahydroisoquinoline may have thestructure:

wherein Z₁₁₂, Z₁₁₃, and Z₁₁₄ are independently hydrogen, hydroxyl,hydrocarbyl, substituted hydrocarbyl, alkoxy, alkenoxy, alkynoxy, oraryloxy. In one particular embodiment in which thetetrahydroisoquinoline has the structure shown above, Z₁₁₂, Z₁₁₃, andZ₁₁₄ are independently hydrogen, hydroxyl, alkyl, substituted alkyl,aryl, substituted aryl, alkoxy, or aryloxy.

In combination, among certain of the preferred embodiments are thioacidscorresponding to Formula (2) wherein Z₁ is heteroaryl, heterocyclo, orhas the formula:

wherein Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄ are independently hydrogen, amino,alkoxy, nitro, or trihalomethoxy (e.g., trifluoromethoxy); or Z₁ is—(CH₂)_(x)—Z₁₀₂ wherein Z₁₀₂ is hydrogen, hydroxyl, protected hydroxyl,heterocyclo, amino, amido, alkoxy, aryloxy, cyano, nitro, thiol, or anacetal, ketal, ester, ether, or thioether, and x is 1, 2, or 3. Stillmore preferably in these embodiments, Z₁ is phenyl or napthyl optionallysubstituted with one or more amino, alkoxy, nitro, or trihalomethoxygroups, or Z₁ is aminoalkyl, or substituted or unsubstituted thiazolyl,furyl, or isoxazolyl.

In certain embodiments, the thioacids (1) are selected from the groupconsisting of (TA1), (TA2), (TA3), (TA4), (TA5), (TA6), (TA7), (TA8),(TA9), and (TA10):

In one particular embodiment, the thioacid (1) corresponds to one ormore of formulae: (TA2), (TA3), (TA4), (TA5), (TA9), and (TA10). Inanother particular embodiment, the thioacid (1) corresponds to one ormore of formulae: (TA2), (TA3), (TA4), (TA5), (TA6), and (TA7).

In general, the thioacids described above for use in the processesdescribed herein are commercially available or can be prepared accordingto conventional organic synthesis techniques.

Sulfonyl Azides

The sulfonyl azides for use in reacting with the thioacids correspondingto Formula (1) in the acylsulfonamide-forming processes described hereingenerally correspond to Formula (2):

wherein Z₂ is hydrocarbyl, substituted hydrocarbyl, heteroaryl, orheterocyclo. Typically, such hydrocarbyl substituents for Z₂ containfrom 1 to 20 carbon atoms and may be linear, branched, or cyclic, andsaid substituted hydrocarbyl, heteroaryl, and heterocyclo moieties forZ₂ may be substituted with one or more of ═O, —OH, —OR_(Z), —COOH,—COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z),—SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo (including F, Cl, Brand I), among others, wherein each occurrence of R_(Z) may behydrocarbyl or substituted hydrocarbyl (e.g., substituted orunsubstituted alkyl, substituted or unsubstituted aryl, or substitutedor unsubstituted aralkyl).

In general, although Z₂ is hydrocarbyl, substituted hydrocarbyl,heteroaryl, or heterocyclo, in certain embodiments Z₂ is substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, alkaryl, or aralkyl. In oneparticular embodiment, Z₂ is aryl or substituted aryl; thus, forexample, Z₂ may have the formula:

wherein Z₂₀, Z₂₁, Z₂₂, Z₂₃, and Z₂₄ are independently hydrogen, halo,hydrocarbyl, substituted hydrocarbyl, alkoxy, alkenoxy, alkynoxy,aryloxy, nitro, cyano, amino, or amido, or two of Z₂₀, Z₂₁, Z₂₂, Z₂₃,and Z₂₄, together with the carbon atoms to which they are attached, forma fused carbocyclic (e.g., napthyl) or heterocyclic ring. In anotherparticular embodiment, Z₂ is phenyl, substituted phenyl, napthyl, orsubstituted napthyl.

In one embodiment in which Z₂ corresponds to the aryl or substitutedaryl structure illustrated above, Z₂₀, Z₂₁, Z₂₂, Z₂₃, and Z₂₄ areindependently alkyl, substituted alkyl, amino, alkoxy, alkenoxy,alkynoxy, or aryloxy. In a particular embodiment, Z₂₀, Z₂₁, Z₂₃, and Z₂₄are hydrogen and Z₂₃ is alkyl, substituted alkyl, amino, alkoxy,alkenoxy, alkynoxy, or aryloxy.

Where one or more of Z₂₀, Z₂₁, Z₂₂, Z₂₃, and Z₂₄ are substituted alkyl,for example, the alkylene moieties may be substituted, for example, with═O, —OH, —OR_(Z), —COOH, —COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z),—NO₂, —SH, —SR_(Z), —SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo(including F, Cl, Br and I), among others, wherein each occurrence ofR_(Z) may be hydrocarbyl or substituted hydrocarbyl (e.g., substitutedor unsubstituted alkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted aralkyl). In one particular embodiment, Z₂corresponds to the aryl or substituted aryl structure illustrated above,wherein Z₂₀, Z₂₁, Z₂₃, and Z₂₄ are hydrogen and Z₂₂ is —N(Z₂₂₀)(Z₂₂₁) or—CH₂—N(Z₂₂₀)(Z₂₂₁), wherein Z₂₂₀ and Z₂₂₁ are independently hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, or Z₂₂₀ and Z₂₂₁together with the nitrogen atom to which they are attached, form asubstituted or unsubstituted alicyclic, bicyclic, aryl, or heterocyclicmoiety. In another particular embodiment, Z₂ corresponds to the aryl orsubstituted aryl structure illustrated above, wherein Z₂₀, Z₂₁, Z₂₃, andZ₂₄ are hydrogen and Z₂₂ is —N(Z₂₂₀)(Z₂₂₁) or —CH₂—N(Z₂₂₀)(Z₂₂₁),wherein Z₂₂₀ and Z₂₂₁ are independently hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, or Z₂₂₀ and Z₂₂₁ together with thenitrogen atom to which they are attached, form a substituted orunsubstituted alicyclic, bicyclic, aryl, or heterocyclic moiety; morepreferably in this embodiment, Z₂ is an N,N-disubstituted (amino)phenylor (aminomethyl)phenyl. Substituents for the Z₂₂₀ and Z₂₂₁ moieties maybe, for example, ═O, —OH, —OR_(Z), —COOH, —COOR_(Z), —CONH₂, —NH₂,—NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z), —SO₂R_(Z), —SO₂H, —SOR_(Z),heterocyclo, and halo (including F, Cl, Br and I), among others, whereineach occurrence of R_(Z) may be hydrocarbyl or substituted hydrocarbyl(e.g., substituted or unsubstituted alkyl, substituted or unsubstitutedaryl, or substituted or unsubstituted aralkyl).

In one particular embodiment in which Z₂ corresponds to the aryl orsubstituted aryl structure illustrated above, Z₂₀, Z₂₁, Z₂₂, Z₂₃, andZ₂₄ are independently alkyl (straight, branched, or cyclic), alkenyl(straight, branched, or cyclic), alkynyl (straight or branched), aryl,alkoxy, arylalkoxy, aryloxy, aryloxyalkoxy, alkylcarbonyloxy,alkylsulfanyl, arylsulfanyl, arylsulfanylalkoxy, cycloalkylalkoxy,cycloalkyloxy, cyano, halo, haloalkyl, haloalkoxy, heterocyclo,(heterocyclo)oxy, nitro, and amino. Where one or more of Z₂₀, Z₂₁, Z₂₂,Z₂₃, and Z₂₄ are amino, the amino moiety may have the formula:—N(Z_(X))(Z_(Y)) wherein Z_(X) and Z_(Y) are independently hydrogen,alkyl, alkenyl, alkoxyalkyl, alkoxycarbonylalkyl, alkylsulfanylalkyl,alkylsulfonylalkyl, aryl, arylalkyl, arylalkylsulfanylalkyl,aryloxyalkyl, arylsulfanylalkyl, arylsulfinylalkyl, arylsulfonylalkyl,carboxyalkyl, cycloalkenyl, cycloalkenylalkyl, cycloalkyl,(cycloalkyl)alkyl, cycloalkylcarbonyl, heterocyclo, (heterocyclo)alkyl,(heterocyclo)sulfanylalkyl, hydroxyalkyl, or a nitrogen protectinggroup, or Z_(X) and Z_(Y), together with the nitrogen atom to which theyare attached, form a substituted or unsubstituted imidazolyl,morpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, pyrrolyl,thiomorpholinyl, or thiomorpholinyl dioxide moiety.

In another particular embodiment, Z₂ is alkyl or substituted alkyl. Inaccordance with this embodiment, therefore, Z₂ may be —(CH₂)_(X)—Z₂₀₀wherein Z₂₀₀ is hydrogen, hydroxyl, protected hydroxyl, heterocyclo,amino, amido, alkoxy, aryloxy, cyano, nitro, thiol, or an acetal, ketal,ester, ether, or thioether, and x is 1, 2, or 3.

Alternatively, Z₂ may be heteroaryl. Thus, for example, Z₂ may besubstituted or unsubstituted furyl, thienyl, pyrrolyl, oxazolyl,imidazolyl, pyridyl, pyrimidyl, purinyl, triazolyl, or thiazolyl.

In another alternative embodiment, Z₂ is heterocyclo. In accordance withthis embodiment, for example, Z₂ may be substituted or unsubstitutedmorpholino, pyran, tetrahydropyran, piperazinyl, piperidinyl,tetrahydropyridinyl, pyrrolidinyl, pyrrolinyl, 1,4-diazepanyl, orazepinyl. In these embodiments, the substituents for the substitutedgroups may correspond to those described above in connection with Z₂₀,Z₂₁, Z₂₂, Z₂₃, and Z₂₄.

In certain embodiments, the sulfonyl azides (2) are selected from thegroup consisting of (SZ1), (SZ2), (SZ3), (SZ4), (SZ5), (SZ6), (SZ7),(SZ8), (SZ9), (SZ10), (SZ11), (SZ12), (SZ13), (SZ14), (SZ15), (SZ16),and (SZ17):

In one particular embodiment, the sulfonyl azide (2) corresponds to oneor more of formulae: (SZ9), (SZ10), (SZ11), (SZ15), (SZ16), and (SZ17).In general, the sulfonyl azides described above for use in the processesdescribed herein are commercially available or can be prepared accordingto conventional organic synthesis techniques.

Bcl-2 Family Proteins

The thioacid (1) and the sulfonyl azide (2), or libraries thereof, arereacted in the presence of a biological target. In general, thebiological target is a biological molecule involved in one or morebiological pathways associated with various diseases and conditionsincluding cancer, diabetes, neurodegenerative diseases, cardiovasculardiseases, respiratory diseases, digestive system diseases, infectiousdiseases, inflammatory diseases, autoimmune diseases, and the like.Likewise, a range of biological pathways may be involved, including cellcycle regulation (e.g., cellular proliferation and apoptosis),angiogenesis, signaling pathways, tumor suppressor pathways,inflammation, oncogenes, and growth factor receptors, among a variety ofothers.

As noted above, the Bcl-2 family of proteins includes bothanti-apoptotic molecules and pro-apoptotic molecules. The anti-apoptoticBcl-2 family members (e.g., Bcl-2, Bcl-X_(L), Mcl-1, A1/BFL-1, Boo/Diva,Bcl-w, and Bcl-y) inhibit the release of certain pro-apoptotic factorsfrom mitochondria, whereas pro-apoptotic Bcl-2 family members (e.g.,Bak, Bax, Bad, tBid, Harakiri (HRK), Bim, Bcl-Xs, Bmf, Egl-1, Puma, andNoxa) induce the release of mitochondrial apoptogenic molecules into thecytosol. In accordance the process described herein, the thioacid(s) (1)is/are reacted with the sulfonyl azide(s) (2) in the presence of aprotein of the Bcl-2 family; thus, in one embodiment the Bcl-2 familyprotein is an anti-apoptotic Bcl-2 family protein, and in anotherembodiment the Bcl-2 family protein is a pro-apoptotic Bcl-2 familyprotein. In some of these embodiments, the Bcl-2 family proteinscontemplated include, but are not limited to, Bcl-2, Bcl-X_(L), Mcl-1,A1/BFL-1, Boo/Diva, Bcl-w, Bcl-y, Bak, Bax, Bad, tBid, Harakiri, Bim,Bcl-Xs, Bmf, Egl-1, Puma, and Noxa. It is also contemplated that two ormore Bcl-2 protein family members may be utilized in the reaction. Inone particular embodiment, the Bcl-2 family protein is Bcl-X_(L). Inanother particular embodiment, the Bcl-2 family protein is Mcl-1.

Acylsulfonamides

The processes described herein generally utilize the biological targetmolecule (e.g., Bcl-X_(L) or Mcl-1) as the reaction vessel or reactiontemplate to assemble an acylsulfonamide compound having preferentialbinding to the biological target, from one or more thioacids and one ormore sulfonyl azides. Thus, the target-guided synthesis strategyutilizes the biological molecule itself as a template for generatingpotential ligand inhibitors from the initial building block fragments orscaffolds (i.e., the thioacids and the sulfonyl azides in the library),that are selectively bound to the target biomolecule and thenirreversibly linked to each other within the confines of the bindingpockets of the target protein. As this approach employs the biologicaltarget to assemble its own inhibitors from relatively few startingreagents (which can be combined in thousands or tens of thousands ofdifferent ways), rather than requiring tedious synthesis, purification,and screening of thousands of library compounds, it is more efficientthan conventional combinatorial chemistry techniques. However, asdescribed in further detail below, certain aspects of combinatorialchemistry can be used in the methods described herein.

The thioacids and the sulfonyl azides generally combine to form anacylsulfonamide. These techniques are capable of producing high-affinityinhibitors by assembling the building block reagents irreversibly insidethe binding pockets of a target biomolecule. Subsequent screening oftarget biomolecule-generated “hits” then establish their bindingaffinity to and specificity for the target. Once the “hit” compounds aredetermined, they can be synthesized according to conventional organicchemistry methods such as described below, or extracted from the targetprotein and purified in trace amounts.

For bivalent molecules that have multiple interactions with the Bcl-2family protein, the resulting hits are very potent (e.g., highaffinity); the bivalent molecules bind to the protein binding site andreach into the substrate pocket. For entropy reasons (e.g., avoidance ofthe loss of three degrees of rotational and translational freedom),among other things, ligand inhibitors display much higher affinity totheir biological targets than the individual components. Thus, eveninitial compound (e.g., thioacids and sulfonyl azides) fragments withonly modest micromolar affinity to individual binding pockets cangenerate nanomolar inhibitors when coupled together to permit optimalbinding interactions with the biological target. Thus, the bindingaffinity of the building block reagent (i.e., scaffold) or precursor tothe Bcl-2 family protein does not need to be in the nanomolar range.

The general approach of in situ ligation chemistry is illustrated inFIG. 2, and ligation chemistry techniques are described, for example, inthe following references: Kolb et al., Angew. Chem. Int. Ed. 2001, 40,2004-2021; Kolb et al., Drug Discovery Today 2003, 8, 1128-1137;Rostovtsev et al., Angew. Chem. Int. Ed. 2002, 41, 2596-2599; Tornoe etal., Journal of Organic Chemistry 2002, 67, 3057-3064; Wang et al.,Journal of the American Chemical Society 2003, 125, 3192-3193; Lee etal., Journal of the American Chemical Society 2003, 125, 9588-9589;Lewis et al., Angew. Chem., Int. Ed. 2002, 41, 1053-1057; Manetsch etal., Journal of the American Chemical Society 2004, 126, 12809-12818;Mocharla et al., Angew. Chem. Int. Ed. 2005, 44, 116-120; Whiting etal., Angew. Chem. 2006, 118, 1463-1467; Whiting et al., Angew. Chem.Int. Ed. Engl. 2006, 45, 1435-1439; and Sharpless et al., Expert Opin.Drug Discovery 2006, 1, 525-538.

In particular, the thioacids and sulfonyl azides corresponding toFormula (1) and (2), respectively, undergo an amidation reaction asillustrated in Reaction Scheme (1) (see also, e.g., Shangguan et al. J.Am. Chem. Soc. 2003, 125, 7754-7755). As noted above, the reaction ofthe thioacid and the sulfonyl azide is templated by the biologicaltarget molecule, a Bcl-2 family protein, in situ within its bindingpockets. Typically, several thioacids (1) and sulfonyl azides (2) in theform of one or more libraries will be reacted in the presence of theBcl-2 family protein; the resulting acylsulfonamide(s) (3) which bind(s)to the Bcl-2 family protein will be the compound(s) of interest (e.g.,for further synthesis, testing, and analysis).

Thus, the acylsulfonamides which can be prepared in accordance with theprocess described herein generally correspond to Formula (3):

wherein Z₁ and Z₂ are as defined in connection with Formulae (1) and(2).

For instance, in one embodiment, Z₁ is aryl, substituted aryl, orheteroaryl. Thus, in certain embodiments. Z₁ has the formula:

wherein

Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄ are independently hydrogen, hydroxyl,protected hydroxyl, halo, hydrocarbyl, substituted hydrocarbyl,heterocyclo, heteroaryl, alkoxy, alkenoxy, alkynoxy, aryloxy, arylalkoxy(heterocyclo)alkoxy, trihaloalkoxy, amino, amido, or cyano, or two ofZ₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄, together with the carbon atoms to whichthey are attached, form a fused carbocyclic (e.g., napthyl) orheterocyclic ring. In one particular embodiment, Z₁₀, Z₁₁, Z₁₂, Z₁₃, andZ₁₄ are independently hydrogen, amino, alkoxy, nitro, or trihalomethoxy.

In an alternative embodiment, Z₁ has the formula:

wherein A is phenyl or a five- or six-membered aromatic carbocyclic orheterocyclic ring wherein from one to three carbon atoms may be replacedby a heteroatom selected from N, O, or S, and wherein A is substitutedwith Z₁₀₀ and Z₁₀₁ through ring carbon atoms or ring heteroatoms, andZ₁₀₀ and Z₁₀₁ are independently hydrogen, hydrocarbyl, substitutedhydrocarbyl, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy,heterocyclo(alkoxy), or halo.

In other embodiments, Z₁ is substituted or unsubstituted furyl, thienyl,pyridyl, oxazolyl, isoxazolyl, imidazolyl, pyridyl, pyrimidyl, purinyl,triazolyl, or thiazolyl, or Z₁ is substituted or unsubstitutedmorpholino, pyran, tetrahydropyran, piperazinyl, piperidinyl,tetrahydropyridinyl, pyrrolidinyl, pyrrolinyl, 1,4-diazepanyl, orazepinyl. In still other embodiments, Z₁ is alkyl or substituted alkyl;here, for example, Z₁ may be —(CH₂)_(x)—Z₁₀₂ wherein Z₁₀₂ is hydrogen,hydroxyl, protected hydroxyl, heterocyclo, amino, amido, alkoxy,aryloxy, cyano, nitro, thiol, or an acetal, ketal, ester, ether, orthioether, and x is 1, 2, or 3. Substituents for such groups in theseembodiments may be selected from the group consisting of ═O, —OH,—OR_(Z), —COOH, —COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂,—SH, —SR_(Z), —SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo(including F, Cl, Br and I), among others, wherein each occurrence ofR_(Z) may be hydrocarbyl or substituted hydrocarbyl (e.g., substitutedor unsubstituted alkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted aralkyl).

Alternatively, Z₁ may be heteroaryl, heterocyclo, or have the formula:

wherein Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄ are independently hydrogen, amino,alkoxy, nitro, or trihalomethoxy (e.g., trifluoromethoxy); or Z₁ may be—(CH₂)_(x)—Z₁₀₂ wherein Z₁₀₂ is hydrogen, hydroxyl, protected hydroxyl,heterocyclo, amino, amido, alkoxy, aryloxy, cyano, nitro, thiol, or anacetal, ketal, ester, ether, or thioether, and x is 1, 2, or 3.

Similarly, in these and other embodiments, Z₂ may be substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, alkaryl, or aralkyl. Thus,in certain embodiments, for example, Z₂ may have the formula:

wherein

Z₂₀, Z₂₁, Z₂₂, Z₂₃, and Z₂₄ are independently hydrogen, halo,hydrocarbyl, substituted hydrocarbyl, alkoxy, alkenoxy, alkynoxy,aryloxy, nitro, cyano, amino, or amido, or two of Z₂₀, Z₂₁, Z₂₂, Z₂₃,and Z₂₄, together with the carbon atoms to which they are attached, forma fused carbocyclic or heterocyclic ring. For instance, Z₂₀, Z₂₁, Z₂₂,Z₂₃, and Z₂₄ may independently be alkyl, substituted alkyl, amino,alkoxy, alkenoxy, alkynoxy, or aryloxy. In another embodiment, Z₂ isphenyl, substituted phenyl, napthyl, or substituted napthyl.

In other embodiments, Z₂ is substituted or unsubstituted furyl, thienyl,pyridyl, oxazolyl, isoxazolyl, imidazolyl, pyridyl, pyrimidyl, purinyl,triazolyl, or thiazolyl, or Z₂ is substituted or unsubstitutedmorpholino, pyran, tetrahydropyran, piperazinyl, piperidinyl,tetrahydropyridinyl, pyrrolidinyl, pyrrolinyl, 1,4-diazepanyl, orazepinyl. In still other embodiments, Z₂ is alkyl or substituted alkyl;here, for example, Z₂ may be —(CH₂)_(x)—Z₂₀₀ wherein Z₂₀₀ is hydrogen,hydroxyl, protected hydroxyl, heterocyclo, amino, amido, alkoxy,aryloxy, cyano, nitro, thiol, or an acetal, ketal, ester, ether, orthioether, and x is 1, 2, or 3.

In combination, Z₁ and Z₂ may each be a substituted or unsubstitutedaryl or heteroaryl moiety. In one particular embodiment, Z₁ is anN,N-disubstituted (aminomethyl)phenyl moiety and Z₂ is apara-substituted benzene or napthyl moiety. The substituents for Z₁and/or Z₂ in this embodiment may be, for example, ═O, —OH, —OR_(Z),—COOH, —COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH,—SR_(Z), —SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo (including F,Cl, Br and I), among others, wherein each occurrence of R_(Z) may behydrocarbyl or substituted hydrocarbyl (e.g., substituted orunsubstituted alkyl, substituted or unsubstituted aryl, or substitutedor unsubstituted aralkyl), among other things.

In one particular embodiment, the acylsulfonamide (3) has the formula:

wherein

Z₁ is:

Z₂ is:

Z₁₁ and Z₁₃ are alkyl, substituted alkyl, —OH, —OR_(Z), —COOH,—COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z),—SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo, among others, whereineach occurrence of R_(Z) is substituted or unsubstituted alkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedaralkyl;

Z₂₂ is —N(Z₂₂₀)(Z₂₂₁) or —CH₂—N(Z₂₂₀)(Z₂₂₁), wherein Z₂₂₀ and Z₂₂₁ areindependently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, or Z₂₂₀ and Z₂₂₁ together with the nitrogen atom to which they areattached, form a substituted or unsubstituted alicyclic, bicyclic, aryl,or heterocyclic moiety; and

Z₁₀, Z₁₂, Z₁₄, Z₂₀, Z₂₁, Z₂₃, and Z₂₄ are hydrogen.

In certain embodiments, the acylsulfonamides (3) are selected from thegroup consisting of (SZ4TA2), (SZ7TA2), (SZ9TA5), (SZ9TA2), (SZ10TA2),(SZ15TA3), (SZ15TA8), (SZ16TA6), (SZ16TA8), (SZ17TA7), (SZ2TA1),(SZ2TA2), (SZ2TA3), (SZ4TA1), (SZ5TA1), (SZ5TA2), (SZ9TA1), (SZ10TA1),(SZ10TA5), (SZ15TA1), (SZ15TA2), (SZ15TA4), (SZ15TA5), (SZ15TA6),(SZ15TA7), (SZ15TA9, (SZ15TA10), (SZ17TA3), (SZ3TA6), (SZ3TA9), and(SZ9TA7):

In one particular embodiment, the acylsulfonamide (3) corresponds to oneor more of formulae: (SZ4TA2), (SZ7TA2), (SZ9TA5), (SZ9TA2), (SZ10TA2),(SZ15TA3), (SZ15TA8), (SZ16TA6), (SZ16TA8), and (SZ17TA7). In anotherparticular embodiment, the acylsulfonamide (3) corresponds to one ormore of formulae: (SZ2TA1), (SZ2TA2), (SZ2TA3), (SZ4TA1), (SZ5TA1),(SZ5TA2), (SZ9TA1), (SZ10TA1), (SZ10TA5), (SZ15TA1), (SZ15TA2),(SZ15TA4), (SZ15TA5), (SZ15TA6), (SZ15TA7), (SZ15TA9, (SZ15TA10),(SZ17TA3), (SZ3TA6), (SZ3TA9), and (SZ9TA7).

Certain other preferred acylsulfonamides are disclosed in U.S. Pat. No.6,720,338 to Augeri et al.; U.S. Pat. No. 7,030,115 to Elmore et al.;and U.S. Pat. No. 7,390,799 to Bruncko et al., each of which is herebyincorporated by reference in its entirety.

Generally, the processes described herein are not wholly dependent onthe screening of final compounds, prepared through traditional means,but rather allow the Bcl-2 family protein to select and combine buildingblocks that fit into its binding site to assemble its own inhibitormolecules. For example, with just 2 to 200 building blocks (1 to 100mono-thioacids and 1 to 100 mono-sulfonyl azides, e.g., in libraries ofcompounds), one can quickly scan through 1 to 10,000 possiblecombinations (1×1 to 100×100) without actually having to make and testthese compounds via conventional synthesis and analysis. This numberbecomes even larger, with the same number of building blocks, if oneincludes di- or tri-thioacids or -sulfonyl azides, thereby providing thetarget protein with greater flexibility to choose the appropriatebuilding block and functional group at the same time. The screeningmethod is as simple as determining whether or not the product has beenformed in a given test mixture by LC/MS, or other suitable instrument. Acompound that is formed by the target Bcl-2 family protein likely to bea good and selective binder, due to the multivalent nature of theinteraction. In one embodiment, 1 to 10 thioacids corresponding toFormula (1) and 1 to 17 sulfonyl azides corresponding to Formula (2) areincubated or reacted in the presence of the Bcl-2 family protein.

Additional aspects, for example, involve screening methods foridentifying a plurality of molecules that exhibit affinity for thebinding site of the target Bcl-2 family protein. A functional groupcapable of participating in a ligation chemistry reaction, such as anthio or azide group, present on the compounds of Formulae (1) and (2),is also attached to the molecule, optionally via a linker. Individualmembers of the resulting plurality of molecules are then mixed with thetarget molecule and individual members of a plurality or library ofcompounds that may exhibit affinity for a substrate binding site of theprotein. The members of the substrate-binding library have beenchemically modified to include a ligation chemistry functional groupcompatible with the functional group of the library of protein-bindingmolecules. Thus, any pair of thioacid and sulfonyl azide compounds, onefrom each library, that exhibits affinity for the binding sites of theprotein will covalently bond via the acylsulfonamide ligation chemistryfunctional groups in situ. The screening process can utilizeconventional screening equipment known in the art such as multi-wellmicrotiter plates.

A mass spectrometer may be used for sequential, automated data analysisof the screening process. Exemplary spectrometer equipment that can beused include the Agilent MSD 1100 SL system, linear ion trap systems(ThermoFinnigan LTQ), quadrupole ion trap (LCQ), or a quadrupoletime-of-flight (QTOF from Waters or Applied Biosystems). Each of theseanalyzers have very effective HPLC interfaces for LC-MS experiments.

In accordance with one embodiment, using the starting precursorfragment, that may be an anchor molecule, discovery can be performed bydesigning small, targeted compound libraries (e.g., less than 100compounds) based on known drugs and/or substrates. These libraries maybe screened using traditional binding assays. The anchor molecules maybe incubated with the Bcl-2 family protein target and small libraries ofcomplementary ligation chemistry reagents or precursors (e.g.,thioacids, if the anchor molecule is a sulfonyl azide, and vice versa).Each reaction mixture may be analyzed by LC/MS to identify products thatare formed by the Bcl-2 protein. Hit validation is performed throughcompetition experiments to demonstrate that the compound is indeedformed by the protein, and binding assays may establish the bindingaffinities of the protein-generated hits.

The thioacids and sulfonyl azides may also include various linkermoieties between the Z₁ substituent and the carbonyl carbon, between thethiol moiety and the carbonyl carbon, between the Z₂ substituent and the—S(═O)₂— moiety, or between the azide moiety and the —S(═O)₂— moiety.The nature and the length of the linker between the two reacting groupsor precursors may be selected to afford compounds with optimal bindingaffinities. Therefore, various types of linkers can be attached to thesubstrate mimics discussed above. This can readily be accomplishedthrough carbon-heteroatom bond-forming reactions, which can involve theazide groups either directly (acylsulfonamide formation) or indirectly(azide reduction, followed by acylation or sulfonylation of theresulting amines), or other synthesis techniques.

Combinatorial Chemistry Approaches

In a combinatorial approach for identifying or optimizingacylsulfonamides and/or the thioacid and sulfonyl azide building blocksfrom which they are prepared, a large compositional space (e.g., ofthioacids, sulfonyl azides, acylsulfonamides, target proteins,buffer(s), or of relative ratios of two or more of the aforementioned)and/or a large reaction condition space (e.g., of temperature, pressure,reaction time, or other parameter(s)) may be rapidly explored bypreparing libraries of thioacids, sulfonyl azides, acylsulfonamides,and/or target proteins and then rapidly screening such libraries. Thelibraries can comprise, for example, the two or more thioacids, two ormore sulfonyl azides, and/or two or more target biomolecules (for use inthe preparation of acylsulfonamides), or two or more acylsulfonamidesresulting from the reactions described above that are varied withrespect to such scaffolds, proteins, and reaction conditions.

Combinatorial approaches for screening a library can include an initial,primary screening, in which initial reaction mixtures or reactionproduct mixtures are rapidly evaluated to provide valuable preliminarydata and, optimally, to identify several “hits,” e.g., particularcandidate materials having characteristics that meet or exceed certainpredetermined metrics (e.g., performance characteristics, desirableproperties, unexpected and/or unusual properties, etc., such as binding,inhibition, and so on). Such metrics may be defined, for example, by thecharacteristics of a known or standard thioacid, sulfonyl azide, targetprotein, acylsulfonamide, synthetic scheme, or binding parameters.Because local performance maxima may exist in compositional spacesbetween those evaluated in the primary screening of the first librariesor alternatively, in process-condition spaces different from thoseconsidered in the first screening, it may be advantageous to screen morefocused libraries (e.g., libraries focused on a smaller range ofcompositional gradients, or libraries comprising compounds havingincrementally smaller structural variations relative to those of theidentified hits) and additionally or alternatively, subject the initialhits to variations in process conditions. Hence, a primary screen can beused reiteratively to explore localized and/or optimized compositionalspace in greater detail. The preparation and evaluation of more focused(thioacid, sulfonyl azide, target protein, or acylsulfonamide) librariescan continue as long as the high-throughput primary screen canmeaningfully distinguish between neighboring library compositions orcompounds.

Once one or more hits have been satisfactorily identified based on theprimary screening, initial scaffold or final product libraries focusedaround the primary-screen hits can be evaluated with a secondary screen,e.g., a screen designed to provide (and typically verified, based onknown materials, to provide) chemical process conditions that relatewith a greater degree of confidence to commercially-important processesand conditions than those applied in the primary screen. For example,certain “real-world-modeling” considerations may be incorporated intothe secondary screen at the expense of methodology speed (e.g., asmeasured by sample throughput) compared to a corresponding primaryscreen. Particular compounds, proteins, reaction conditions, orpost-synthesis processing conditions having characteristics that surpassthe predetermined metrics for the secondary screen may then beconsidered to be “leads.” If desired, additional thioacid, sulfonylazide, acylsulfonamide, or other libraries focused about such leadmaterials can be screened with additional secondary screens or withtertiary screens. Identified lead thioacids, sulfonyl azides,acylsulfonamides, proteins, and/or reaction conditions may besubsequently developed for commercial applications through traditionalbench-scale and/or pilot scale experiments.

While the concept of primary screens and secondary screens as outlinedabove provides a valuable combinatorial research model for investigatingthioacid/sulfonyl azide/acylsulfonamide/Bcl-2 family protein reactions,a secondary screen may not be necessary for certain chemical processeswhere primary screens provide an adequate level of confidence as toscalability and/or where market conditions warrant a direct developmentapproach. Similarly, where optimization of materials having knownproperties of interest is desired, it may be appropriate to start with asecondary screen. In general, the systems, devices and methods, and thebuilding block or final compounds described herein may be applied aseither a primary or a secondary screen, depending on the specificresearch program and goals thereof.

According to certain aspects, methods, systems and devices are disclosedthat improve the efficiency and/or effectiveness of the steps necessaryto characterize a thioacid or sulfonyl azide sample or a plurality ofthioacid or sulfonyl azide samples, or an acylsulfonamide sample or aplurality of acylsulfonamide samples (e.g., libraries of initial andfinal product mixtures comprising the thioacids and sulfonyl azides, andthe acylsulfonamides, respectively). In certain preferred embodiments, aproperty of a plurality of samples or of components thereof can bedetected in a characterization system with an average sample-throughputsufficient for an effective combinatorial or TGS research program. Theproperty may be, for example, protein binding, protein inhibition, orother related or unrelated parameter.

Characterizing a (building block and/or final) sample can include (i)preparing the sample (e.g., synthesis or dilution), (ii) injecting thesample into a mobile phase of a flow characterization system (e.g.,liquid chromatography system, flow-injection analysis system, or relatedapparatus), (iii) separating the sample chromatographically, (iv)detecting a property of the sample or of one or more components thereof,and/or (v) correlating the detected property or parameter to acharacterizing property or parameter of interest. Variouscharacterization protocols may be employed involving some or all of theaforementioned steps. For example, a property of a thioacid, sulfonylazide, or resulting acylsulfonamide sample (or libraries thereof) may bedetected in a non-flow, static system either with preparation (steps (i)and (iv)) or without preparation (step (iv)). Alternatively, a propertyof a sample may be detected in a flow characterization system, eitherwith or without sample preparation and either with or withoutchromatographic separation. In certain characterization protocolsinvolving flow characterization systems without chromatographic analysisor separation, for example, a property of a sample may be detected in aflow-injection analysis system either with preparation (steps (i), (ii),and (iv)) or without preparation (steps (ii) and (iv)). Ifchromatographic separation of a sample is desired, a property of thesample may be detected in a liquid chromatography system either withpreparation (steps (i), (ii), (iii), and (iv)) or without preparation(steps (ii), (iii), and (iv)). While the physically-detected property(e.g., refracted light, absorbed light, scattered light) from twosamples being screened could be compared directly, in most cases thedetected property is preferably correlated to a characterizing propertyof interest (e.g., molecular weight, protein binding, inhibition, etc.)(step (v)).

A plurality of samples may be characterized as described above. As ageneral approach for improving the sample throughput for a plurality ofthioacids, sulfonyl azides, acylsulfonamides, or proteins, each of thesteps, applicable to a given characterization protocol can be optimizedwith respect to time and quality of information, both individually andin combination with each other. Additionally or alternatively, each orsome of such steps can be effected in a rapid-serial, parallel,serial-parallel or hybrid parallel-serial manner, as understood inaccordance with conventional combinatorial chemistry protocols.

The throughput of a plurality of samples through a single step in acharacterization process is improved by optimizing the speed of thatstep, while maintaining, to the extent necessary, theinformation-quality aspects of that step. In many cases, such as withchromatographic or mass spectroscopic analysis, speed can be gained atthe expense of resolution of the separated or analyzed components.Although conventional research norms, developed in the context in whichresearch was rate-limited primarily by the synthesis of samples, mayfind such an approach less than wholly satisfactory, the degree of rigorcan be entirely satisfactory for a primary or a secondary screen of acombinatorial library of samples. For combinatorial research (and aswell, for many on-line process control systems), the quality ofinformation should be sufficiently rigorous to provide forscientifically acceptable distinctions between the compounds or processconditions being investigated, and for a secondary screen, to providefor scientifically acceptable correlation (e.g., values or, for somecases, trends) with more rigorous, albeit more laborious andtime-consuming traditional characterization approaches.

The throughput of a plurality of samples through a series of steps,where such steps are repeated for the plurality of samples, can also beoptimized. In accordance with one approach, one or more steps of thecycle can be compressed relative to traditional approaches or can haveupstream or downstream aspects truncated to allow other steps of thesame cycle to occur sooner compared to the cycle with traditionalapproaches. In another approach, the earlier steps of a second cycle canbe performed concurrently with the later steps of a first cycle. In arapid-serial approach for characterizing a sample, for instance, samplepreparation for a second sample in a series can be effected while thefirst sample in the series is being synthesized, detected, and/oranalyzed. As another example, a second sample in a series can beinjected while the first sample in the series is being synthesized,detected, and/or analyzed.

A characterization protocol for a plurality of samples can involve asingle-step process. In a rapid-serial detection approach for asingle-step process, the plurality of samples and a single detector areserially positioned in relation to each other for serial detection ofthe samples. In a parallel detection approach, two or more detectors areemployed to detect a property of two or more samples simultaneously. Ina direct, non-flow detection protocol, for example, two or more samplesand two or more detectors can be positioned in relation to each other todetect a property of the two or more samples simultaneously. In aserial-parallel detection approach, a property of a larger number ofsamples (e.g., three, four, or more) is detected as follows. First, aproperty of a subset of the three, four, or more samples (e.g., 2samples) is detected in parallel for the subset of samples, and thenserially thereafter, a property of another subset of four or moresamples is detected in parallel.

For characterization protocols involving more than one step (e.g., twoor more of steps (i), (ii), (iii), (iv), and (v), above), optimizationapproaches to effect high-throughput characterization of thioacids,sulfonyl azides, target biomolecules, and resulting acylsulfonamides)can vary. For instance, a plurality of samples can be characterized witha single characterization system (A) in a rapid-serial approach in whicheach of the plurality of samples (A₁, A₂, A₃ . . . A_(n)) are processedserially through the characterization system (A) with each of the steps((i), (ii), (iii), (iv), and (v)) effected in series on each of the ofsamples to produce a serial stream of corresponding characterizingproperty data (d₁, d₂, d₃ . . . d_(n)). This approach benefits fromrelatively minimal capital investment, and may provide sufficientthroughput, particularly when the steps (i), (ii), (iii), (iv), and (v)have been optimized with respect to speed and quality of information. Asanother example, a plurality of samples can be characterized with two ormore characterization systems (A, B, C, D . . . N) in a pure parallel(or for larger libraries, serial-parallel) approach in which theplurality of samples (A₁, A₂, A₃ . . . A_(n)) or a subset thereof areprocessed through the two or more characterization systems (A, B, C, D .. . ZZ) in parallel, with each individual system effecting each step onone of the samples to produce the characterizing property information(A₁, A₂, A₃ . . . A_(n); B₁, B₂, B₃ . . . B_(n); C₁, C₂, C₃ . . . C_(n),etc.) in parallel. This approach is advantageous with respect to overallthroughput, but may be constrained by the required capital investment.

In a hybrid approach, certain of the steps of the characterizationprocess can be effected in parallel, while certain other steps can beeffected in series. Preferably, for example, it may be desirable toeffect the longer, throughput-limiting steps in parallel for theplurality of samples, while effecting the faster, less limiting steps inseries. Such a parallel-series hybrid approach can be exemplified, byparallel sample preparation (step (i)) of a plurality of thioacid,sulfonyl azide, or acylsulfonamide samples (A₁, A₂, A₃ . . . A_(n)),followed by serial injection, chromatographic analysis, detection andcorrelation (steps (ii), (iii), (iv), and (v)) with a singlecharacterization system (A) to produce a serial stream of correspondingcharacterizing property information (d₁, d₂, d₃ . . . d_(n)). In anotherexemplary parallel-series hybrid approach, a plurality of thioacid,sulfonyl azide, or acylsulfonamide samples (A₁, A₂, A₃ . . . A_(n)) areprepared, reacted, and injected in series into the mobile phase of fouror more characterizing systems (e.g., LC/MS) (A, B, C . . . ZZ), andthen detected and correlated in a slightly offset (staggered) parallelmanner to produce the characterizing property information (d₁, d₂, d₃ .. . d_(n)) in the same staggered-parallel manner. If each of the systemshas the same processing rates, then the extent of the parallel offset(or staggering) will be primarily determined by the speed of the serialpreparation and reaction. In a variation of the preceding example, wherethe detection and correlation steps are sufficiently rapid, a pluralityof thioacid, sulfonyl azide, or acylsulfonamide samples (A₁, A₂, A₃ . .. A_(n)) could be characterized by serial sample preparation andreaction, staggered-parallel analysis, and then serial correlation, toproduce the characterizing property information (d₁, d₂, d₃ . . . d_(n))in series. In this case, the rate of injection into the variousseparation columns is preferably synchronized with the rate ofdetection. In general, optimization of individual characterization steps(e.g., steps (i), (ii), (iii), (iv), and (v)) with respect to speed andquality of information can improve sample throughput regardless ofwhether the overall characterization scheme involves a rapid-serial orparallel aspect (i.e., true parallel, serial-parallel or hybridparallel-series approaches).

A plurality or library of samples generally comprises 2 or morethioacid, sulfonyl azide, target protein, or acylsulfonamide samples.The individual compounds may be physically or temporally separated fromeach other, e.g., by residing in different sample containers, by havinga membrane or other partitioning material positioned between samples, bybeing partitioned (e.g., in-line) with an intervening fluid, by beingtemporally separated in a flow process line (e.g., as sampled forprocess control purposes), or otherwise, or two, three, or more compoundsamples may be combined or otherwise reside in the same samplecontainer. In certain embodiments, the plurality (or library) of samplestypically comprises 4 or more samples (e.g., 4 or more differentthioacid, sulfonyl azide, or acylsulfonamide compounds), while incertain other embodiments, 8 or more samples (e.g., 4 or more differentthioacid, sulfonyl azide, or acylsulfonamide compounds). Four samplescan be employed, for example, in connection with experiments having onecontrol sample and three samples varying (e.g., with respect tocompound, target, or process conditions as discussed above) to berepresentative of a high, a medium and a low-value of the varied factor,and thereby, to provide some indication as to trends. Four samples mayalso be a minimum number of samples to effect a serial-parallelcharacterization approach, as described above (e.g., with twodetector/analyzers operating in parallel). Eight samples can provide foradditional variations in the explored factor space. Higher numbers ofsamples and libraries thereof can be investigated, according to themethods described herein, to provide additional insights into largercompositional and/or process space. In some cases, for example, theplurality of samples can be 15 or more samples, 20 or more samples, 40or more samples, 80 or more samples, or more. Such numbers can beloosely associated with standard configurations of other parallelreactor configurations and/or of standard sample containers (e.g.,96-well microtiter plate-type formats). Moreover, even larger numbers ofsamples can be characterized according to the methods described hereinfor larger scale research endeavors. Hence, the number of thioacid,sulfonyl azide, and acylsulfonamide samples prepared and analyzed can be150 or more, 400 or more, 500 or more, 750 or more, 1,000 or more, 1,500or more, 2,000 or more, 5,000 or more and 10,000 or more. As such, thenumber of samples can range from about 2 samples to about 10,000samples, or more, and preferably from about 8 samples to about 10,000samples, or more. In some cases, in which processing of samples usingtypical 96-well microtiter-plate formatting is convenient or otherwisedesirable, the number of samples can be 96*N, where N is an integerranging from about 1 to about 100. For many applications, N can suitablyrange from 1 to about 20, and in some cases, from 1 to about 7.

The plurality of samples can likewise be a library of samples, e.g., alibrary of thioacids, a library of sulfonyl azides, and/or a library ofacylsulfonamides. A library of samples generally comprises an array oftwo or more different thioacid, sulfonyl azide, and/or acylsulfonamidesamples spatially separated, e.g., on a common substrate, or temporallyseparated, e.g., in a flow system. Candidate samples (i.e., members)within a library may differ in a definable and typically predefined way,including with regard to chemical structure (i.e., the substituents onthe thioacid or sulfonyl azide), processing (e.g., synthesis) history(including the biological target utilized in the target-guidedsynthesis), mixtures of interacting components, purity, etc. The samplesmay be spatially separated, for instance, at an exposed surface of thesubstrate, such that the array of samples are separately addressable forcharacterization thereof. The two or more different samples can residein sample containers formed as wells in a surface of the substrate. Thenumber of samples included within the library can generally be the sameas the number of samples included within the plurality of samples, asdiscussed above. In general, however, not all of the samples within alibrary of samples need to be different samples. When process conditionsare to be evaluated, the libraries may contain only one type of sample.Typically, however, for combinatorial research applications, at leasttwo or more, preferably at least four or more, even more preferablyeight or more and, in many cases most, and allowably each of theplurality of samples in a given library of samples will be differentfrom each other. Specifically, a different sample can be included withinat least about 50%, preferably at least 75%, preferably at least 80%,even more preferably at least 90%, still more preferably at least 95%,yet more preferably at least 98% and most preferably at least 99% of thesamples included in the sample library. In some cases, all of thesamples in a library of samples will be different from each other.

In general, the substrate can be a structure having a rigid orsemi-rigid surface on which or into which the array of samples can beformed or deposited. The substrate can be of any suitable material, andpreferably consists essentially of materials that are inert with respectto the samples of interest (including, for example, the thioacid,sulfonyl azide, acylsulfonamide, or the biological target molecule(e.g., the Bcl-2 family protein(s)). Certain materials will, therefore,be less desirably employed as a substrate material for certain reactionprocess conditions (e.g., high temperatures or high pressures) and/orfor certain reaction mechanisms. Stainless steel, silicon, includingpolycrystalline silicon, single-crystal silicon, sputtered silicon, andsilica (SiO₂) in any of its forms (quartz, glass, etc.), for example,may be substrate materials. Other known materials (e.g., siliconnitride, silicon carbide, metal oxides (e.g., alumina), mixed metaloxides, metal halides (e.g., magnesium chloride), minerals, zeolites,and ceramics) may also be suitable for a substrate material in someapplications. Organic and inorganic polymers may also be suitablyemployed in some applications. Exemplary polymeric materials that can besuitable as a substrate material in particular applications includepolystyrenes, polyimides such as Kapton™, polypropylene,polytetrafluoroethylene (PTFE) and/or polyether etherketone (PEEK),among others. The substrate material is also preferably selected forsuitability in connection with known fabrication techniques. As to form,the sample containers formed in, at or on a substrate can be preferably,but are not necessarily, arranged in a substantially flat, substantiallyplanar surface of the substrate. The sample containers can be formed ina surface of the substrate as dimples, wells, raised regions, trenches,or the like. Non-conventional substrate-based sample containers, such asrelatively flat surfaces having surface-modified regions (e.g.,selectively wettable regions) can also be employed. The overall sizeand/or shape of the substrate is not limiting. The size and shape can bechosen, however, to be compatible with commercial availability, existingfabrication techniques, and/or with known or later-developed automationtechniques, including automated sampling and automatedsubstrate-handling devices, as well as detection and analysis equipment.The substrate is also preferably sized to be portable by humans. Thesubstrate can be thermally insulated if needed, for example, forhigh-temperature and/or low-temperature applications. In preferredembodiments, the substrate is designed such that the individuallyaddressable regions of the substrate can act as reaction vessels forpreparing the acylsulfonamides from the reaction of the thioacids andthe sulfonyl azides in the presence of the biological target (e.g., aBcl-2 protein) in a product mixture (as well as sample containers forthe samples during subsequent characterization thereof). Glass-lined,96-well, 384-well and 1536-well microtiter-type plates, fabricated fromstainless steel, aluminum, composite, polystyrene or other polymers orplastics, may be used as substrates for a library of samples. The choiceof an appropriate specific substrate material and/or form for certainapplications will be apparent to those of skill in the art in view ofthe guidance provided herein.

The library of materials can be a combinatorial library of buildingblocks (e.g., thioacids, sulfonyl azides) or a combinatorial library ofproduct mixtures (e.g., acylsulfonamides). Thioacid libraries cancomprise, for example, a variety of thioacids corresponding to Formula(1) to be used in the target-guided synthesis approaches describedherein. Similarly, sulfonyl azide libraries can comprise, for example, avariety of sulfonyl azides corresponding to Formula (2) to be used inthe target-guided synthesis approaches described herein. Acylsulfonamidelibraries can comprise, for example, product mixtures resulting fromsuch reactions of thioacids and sulfonyl azides (including librariesthereof) that are varied with respect to, for example, particularsubstituent patterns, buffers, biological targets, the relative amountsof components, reaction conditions (e.g., pH, temperature, pressure,reaction time) or any other factor that may affect the reaction. Designvariables for reactions are well known in the art. A library ofthioacid/sulfonyl azide/acylsulfonamide samples may be prepared inarrays, in parallel reactors or in a serial fashion. In certainembodiments, the libraries can be characterized directly, without beingisolated, from the reaction vessel in which the compound(s) wassynthesized.

While such methods may be generally preferred for a combinatorialapproach to lead compound research, they are to be considered exemplaryand non-limiting. As noted above, the particular samples characterizedaccording to the methods and with the apparatus disclosed herein can befrom any source, including, but not limited to product mixturesresulting from combinatorial synthesis approaches or from target-guidedsynthesis approaches.

Pharmaceutical Compositions and Methods for Treatment

Other aspects involve methods for treatment of various conditions anddiseases using the compounds described herein. According to methods oftreatment, the compounds described herein, and particularly theacylsulfonamides corresponding to Formula (3) can be useful for theprevention of metastases from the tumors described above either whenused alone or in combination with radiotherapy and/or otherchemotherapeutic treatments conventionally administered to patients fortreating cancer. When using the compounds for chemotherapy, for example,the specific therapeutically effective dose level for any particularpatient will depend upon factors such as the disorder being treated andthe severity of the disorder; the activity of the particular compoundused; the specific compound employed; the age, body weight, generalhealth, sex, and diet of the patient; the time of administration; theroute of administration; the rate of excretion of the compound employed;the duration of treatment; and drugs used in combination with orcoincidentally with the compound used. For example, when used in thetreatment of solid tumors, the compounds can be administered withchemotherapeutic agents such as alpha interferon, COMP(cyclophosphamide, vincristine, methotrexate, and prednisone),etoposide, mBACOD (methotrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine, and dexamethasone), PRO-MACE/MOPP(prednisone, methotrexate (w/leucovin rescue), doxorubicin,cyclophosphamide, paclitaxel, docetaxel, etoposide/mechlorethamine,vincristine, prednisone, and procarbazine), vincristine, vinblastine,angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG, andthe like. For example, a tumor may be treated conventionally withsurgery, radiation or chemotherapy and a compound disclosed hereinsubsequently administered to extend the dormancy of micrometastases andto stabilize and inhibit the growth of any residual primary tumor.

Additional aspects include compounds which have been described in detailhereinabove or to pharmaceutical compositions which comprise aneffective amount of one or more compounds according to the disclosure,optionally in combination with a pharmaceutically acceptable carrier,additive or excipient (described in further detail below).

Dosage and Amount and Time Course of Treatment

The dose or amount of pharmaceutical compositions including theacylsulfonamide compositions described above administered to the mammalshould be an effective amount for the intended purpose, i.e., treatment(or prophylaxis) of one or more of the diseases, pathological disorders,and medical conditions noted above. Generally speaking, the effectiveamount of the composition administered to the mammal can vary accordingto a variety of factors such as, for example, the age, weight, sex,diet, route of administration, and the medical condition of the mammal.Specifically preferred doses are discussed more fully below. It will beunderstood, however, that the total daily usage of the compositionsdescribed herein will be decided by the attending physician orveterinarian within the scope of sound medical judgment.

The specific therapeutically effective dose level for any particularmammal will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound(s) employed; the age, body weight, general health, sex and dietof the patient; the time of administration; the route of administration;the rate of excretion of the specific compound(s) employed; the durationof the treatment; drugs used in combination or coincidental with thespecific compound(s) employed and like factors well known in the medicaland/or veterinary arts. For example, it is well within the skill of theart to start doses of the compound(s) at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. If desired,the effective daily doses may be divided into multiple doses forpurposes of administration. Consequently, single dose compositions maycontain such amounts or submultiples to make up the daily dose.

Administration of the pharmaceutical composition can occur as a singleevent or over a time course of treatment. For example, one or more ofthe compositions can be administered hourly (e.g., every hour, every twohours, every three hours, every four hours, every five hours, every sixhours, and so on), daily, weekly, bi-weekly, or monthly. For treatmentof acute conditions, the time course of treatment may be at leastseveral hours or days. Certain conditions could extend treatment fromseveral days to several weeks. For example, treatment could extend overone week, two weeks, or three weeks. For more chronic conditions,treatment could extend from several weeks to several months, a year ormore, or the lifetime of the mammal in need of such treatment.Alternatively, the compositions can be administered hourly, daily,weekly, bi-weekly, or monthly, for a period of several weeks, months,years, or over the lifetime of the mammal as a prophylactic measure.

One or more of the compounds may be utilized in a pharmaceuticallyacceptable carrier, additive or excipient at a suitable dose rangingfrom about 0.05 to about 200 mg/kg of body weight per day, preferablywithin the range of about 0.1 to 100 mg/kg/day, most preferably in therange of 0.25 to 50 mg/kg/day. As noted above, the desired dose mayconveniently be presented in a single dose or as divided dosesadministered at appropriate intervals, for example as two, three, fouror more sub-doses per day.

Ideally, the active ingredient should be administered to achieveeffective peak plasma concentrations of the active compound within therange of from about 0.05 uM to about 5 uM. This may be achieved, forexample, by the intravenous injection of about a 0.05 to 10% solution ofthe active ingredient, optionally in saline, or orally administered as abolus containing about 1 mg to about 5 g, preferably about 5 mg to about500 mg of the active ingredient, depending upon the active compound andits intended target. Desirable blood levels may be maintained by acontinuous infusion to preferably provide about 0.01 mg/kg/hour to about2.0 mg/kg/hour or by intermittent infusions containing about 0.05 mg/kgto about 15 mg/kg of the active ingredient. Oral dosages, whereapplicable, will depend on the bioavailability of the compositions fromthe GI tract, as well as the pharmacokinetics of the compositions to beadministered. While it is possible that, for use in therapy, one or morecompositions of the invention may be administered as the raw chemical,it is preferable to present the active ingredient as a pharmaceuticalformulation, presented in combination with a pharmaceutically acceptablecarrier, excipient, or additive.

Routes of Administration, Formulations/Pharmaceutical Compositions

As noted above, the above-described compounds may be dispersed in apharmaceutically acceptable carrier prior to administration to themammal. The carrier, also known in the art as an excipient, vehicle,auxiliary, adjuvant, or diluent, is typically a substance which ispharmaceutically inert, confers a suitable consistency or form to thecomposition, and does not diminish the efficacy of the compound. Thecarrier is generally considered to be “pharmaceutically orpharmacologically acceptable” if it does not produce an unacceptablyadverse, allergic or other untoward reaction when administered to amammal, especially a human.

The selection of a pharmaceutically acceptable carrier will also, inpart, be a function of the route of administration. In general, thecompositions can be formulated for any route of administration so longas the blood circulation system is available via that route. Forexample, suitable routes of administration include, but are not limitedto, oral, parenteral (e.g., intravenous, intraarterial, subcutaneous,rectal, subcutaneous, intramuscular, intraorbital, intracapsular,intraspinal, intraperitoneal, or intrasternal), topical (nasal,transdermal, intraocular), intravesical, intrathecal, enteral,pulmonary, intralymphatic, intracavital, vaginal, transurethral,intradermal, aural, intramammary, buccal, orthotopic, intratracheal,intralesional, percutaneous, endoscopical, transmucosal, sublingual andintestinal administration.

Pharmaceutically acceptable carriers for use in combination with theacylsulfonamide compounds are well known to those of ordinary skill inthe art and are selected based upon a number of factors: the particularcompound used, and its concentration, stability and intendedbioavailability; the subject, its age, size and general condition; andthe route of administration. Suitable nonaqueous,pharmaceutically-acceptable polar solvents include, but are not limitedto, alcohols (e.g., α-glycerol formal, β-glycerol formal,1,3-butyleneglycol, aliphatic or aromatic alcohols having 2 to 30 carbonatoms such as methanol, ethanol, propanol, isopropanol, butanol,t-butanol, hexanol, octanol, amylene hydrate, benzyl alcohol, glycerin(glycerol), glycol, hexylene glycol, tetrahydrofurfuryl alcohol, laurylalcohol, cetyl alcohol, or stearyl alcohol, fatty acid esters of fattyalcohols such as polyalkylene glycols (e.g., polypropylene glycol,polyethylene glycol), sorbitan, sucrose and cholesterol); amides (e.g.,dimethylacetamide (DMA), benzyl benzoate DMA, dimethylformamide,N-(β-hydroxyethyl)-lactamide, N,N-dimethylacetamide amides,2-pyrrolidinone, 1-methyl-2-pyrrolidinone, or polyvinylpyrrolidone);esters (e.g., 1-methyl-2-pyrrolidinone, 2-pyrrolidinone, acetate esterssuch as monoacetin, diacetin, and triacetin, aliphatic or aromaticesters such as ethyl caprylate or octanoate, alkyl oleate, benzylbenzoate, benzyl acetate, dimethylsulfoxide (DMSO), esters of glycerinsuch as mono, di-, or tri-glyceryl citrates or tartrates, ethylbenzoate, ethyl acetate, ethyl carbonate, ethyl lactate, ethyl oleate,fatty acid esters of sorbitan, fatty acid derived PEG esters, glycerylmonostearate, glyceride esters such as mono, di-, or tri-glycerides,fatty acid esters such as isopropyl myristrate, fatty acid derived PEGesters such as PEG-hydroxyoleate and PEG-hydroxystearate,N-methylpyrrolidinone, pluronic 60, polyoxyethylene sorbitol oleicpolyesters such as poly(ethoxylated)₃₀₋₆₀ sorbitol poly(oleate)₂₋₄,poly(oxyethylene)₁₅₋₂₀ monooleate, poly(oxyethylene)₁₅₋₂₀ mono12-hydroxystearate, and poly(oxyethylene)₁₅₋₂₀ mono ricinoleate,polyoxyethylene sorbitan esters such as polyoxyethylene-sorbitanmonooleate, polyoxyethylene-sorbitan monopalmitate,polyoxyethylene-sorbitan monolaurate, polyoxyethylene-sorbitanmonostearate, and Polysorbate® 20, 40, 60 or 80 from ICI Americas,Wilmington, Del., polyvinylpyrrolidone, alkyleneoxy modified fatty acidesters such as polyoxyl 40 hydrogenated castor oil and polyoxyethylatedcastor oils (e.g., Cremophor® EL solution or Cremophor® RH 40 solution),saccharide fatty acid esters (i.e., the condensation product of amonosaccharide (e.g., pentoses such as ribose, ribulose, arabinose,xylose, lyxose and xylulose, hexoses such as glucose, fructose,galactose, mannose and sorbose, trioses, tetroses, heptoses, andoctoses), disaccharide (e.g., sucrose, maltose, lactose and trehalose)or oligosaccharide or mixture thereof with a C₄ to C₂₂ fatty acid(s)(e.g., saturated fatty acids such as caprylic acid, capric acid, lauricacid, myristic acid, palmitic acid and stearic acid, and unsaturatedfatty acids such as palmitoleic acid, oleic acid, elaidic acid, erucicacid and linoleic acid)), or steroidal esters); alkyl, aryl, or cyclicethers having 2 to 30 carbon atoms (e.g., diethyl ether,tetrahydrofuran, dimethyl isosorbide, diethylene glycol monoethylether); glycofurol (tetrahydrofurfuryl alcohol polyethylene glycolether); ketones having 3 to 30 carbon atoms (e.g., acetone, methyl ethylketone, methyl isobutyl ketone); aliphatic, cycloaliphatic or aromatichydrocarbons having 4 to 30 carbon atoms (e.g., benzene, cyclohexane,dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane,sulfolane, tetramethylenesulfon, tetramethylenesulfoxide, toluene,dimethylsulfoxide (DMSO), or tetramethylenesulfoxide); oils of mineral,vegetable, animal, essential or synthetic origin (e.g., mineral oilssuch as aliphatic or wax-based hydrocarbons, aromatic hydrocarbons,mixed aliphatic and aromatic based hydrocarbons, and refined paraffinoil, vegetable oils such as linseed, tung, safflower, soybean, castor,cottonseed, groundnut, rapeseed, coconut, palm, olive, corn, corn germ,sesame, persic and peanut oil and glycerides such as mono-, di- ortriglycerides, animal oils such as fish, marine, sperm, cod-liver,haliver, squalene, squalane, and shark liver oil, oleic oils, andpolyoxyethylated castor oil); alkyl or aryl halides having 1 to 30carbon atoms and optionally more than one halogen substituent; methylenechloride; monoethanolamine; petroleum benzin; trolamine; omega-3polyunsaturated fatty acids (e.g., alpha-linolenic acid,eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid);polyglycol ester of 12-hydroxystearic acid and polyethylene glycol(Solutol® HS-15, from BASF, Ludwigshafen, Germany); polyoxyethyleneglycerol; sodium laurate; sodium oleate; or sorbitan monooleate.

Other pharmaceutically acceptable solvents for use in the invention arewell known to those of ordinary skill in the art, and are identified inThe Chemotherapy Source Book (Williams & Wilkens Publishing), TheHandbook of Pharmaceutical Excipients, (American PharmaceuticalAssociation, Washington, D.C., and The Pharmaceutical Society of GreatBritain, London, England, 1968), Modern Pharmaceutics, (G. Banker etal., eds., 3d ed.) (Marcel Dekker, Inc., New York, N.Y., 1995), ThePharmacological Basis of Therapeutics, (Goodman & Gilman, McGraw HillPublishing), Pharmaceutical Dosage Forms, (H. Lieberman et al., eds.)(Marcel Dekker, Inc., New York, N.Y., 1980), Remington's PharmaceuticalSciences (A. Gennaro, ed., 19th ed.) (Mack Publishing, Easton, Pa.,1995), The United States Pharmacopeia 24, The National Formulary 19,(National Publishing, Philadelphia, Pa., 2000), and A. J. Spiegel etal., Use of Nonaqueous Solvents in Parenteral Products, Journal ofPharmaceutical Sciences, Vol. 52, No. 10, pp. 917-927 (1963).

Formulations containing the above acylsulfonamide compounds may take theform of solid, semi-solid, lyophilized powder, or liquid dosage formssuch as, for instance, aerosols, capsules, creams, emulsions, foams,gels/jellies, lotions, ointments, pastes, powders, soaps, solutions,sprays, suppositories, suspensions, sustained-release formulations,tablets, tinctures, transdermal patches, and the like, preferably inunit dosage forms suitable for simple administration of precise dosages.

Salts and Prodrugs

As noted above, the pharmaceutical compositions may includeacylsulfonamide compounds in their salt form. Typically, the salt willbe a pharmaceutically acceptable salt; that is, a salt prepared frompharmaceutically acceptable non-toxic acids, including inorganic acidsand organic acids. Suitable non-toxic acids include inorganic andorganic acids of basic residues such as amines, for example, acetic,benzenesulfonic, benzoic, amphorsulfonic, citric, ethenesulfonic,fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic,lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,pantothenic, phosphoric, succinic, sulfuric, barbaric acid,p-toluenesulfonic and the like; and alkali or organic salts of acidicresidues such as carboxylic acids, for example, alkali and alkalineearth metal salts derived from the following bases: sodium hydride,sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminumhydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide,ammonia, trimethylammonia, triethylammonia, ethylenediamine, lysine,arginine, ornithine, choline, N,N″-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, n-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like. Pharmaceutically acceptablesalts of the compounds described herein can be prepared by reacting thefree acid or base forms of these compositions with a stoichiometricamount of the appropriate base or acid in water or in an organicsolvent, or in a mixture of the two; generally, nonaqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, each of which is hereby incorporated by reference herein.

Since prodrugs are known to enhance numerous desirable pharmaceuticals(e.g., solubility, bioavailability, manufacturing), the compound(s) maybe delivered in prodrug form. Thus, the present disclosure is intendedto cover prodrugs of the compounds (e.g., acylsulfonamides) describedabove, methods of delivering the same and compositions containing them.Prodrugs generally include any covalently bonded carriers which releasean active parent drug in vivo when such prodrug is administered to amammalian subject. Prodrugs are generally prepared by modifyingfunctional groups present in the compound in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound. Prodrugs include compounds wherein a hydroxyl oramino group is bonded to any group that, when the prodrug isadministered to a mammalian subject, cleaves to form a free hydroxyl orfree amino group, respectively. Examples of prodrugs include, but arenot limited to, acetate, formate, and benzoate derivatives of alcoholand amine functional groups in the compounds and conjugates disclosedherein. Prodrugs of the compound are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals with undue toxicity, irritation, allergic response, andthe like, commensurate with a reasonable benefit/risk ratio, andeffective for their intended use, as well as the zwitterionic forms,where possible, of the compositions of the invention. Prodrugs may referto compounds that are rapidly transformed in vivo to yield thecompound(s) above, for example by hydrolysis in blood. A thoroughdiscussion of prodrugs is provided in the following: Design of Prodrugs,H. Bundgaard, ea., Elsevier, 1985; Methods in Enzymology, K. Widder etal, Ed., Academic Press, 42, p. 309-396, 25 1985; A Textbook of DrugDesign and Development, Krogsgaard-Larsen and H. Bundgaard, ea., Chapter5; “Design and Applications of Prodrugs” p. 113-191, 1991; Advanced DrugDelivery Reviews, H. Bundgard, 8, p. 1-38, 1992; Journal ofPharmaceutical Sciences, 77, p. 285, 30 1988; Chem. Pharm. Bull., N.Nakeya et al, 32, p. 692, 1984; Pro-drugs as Novel Delivery Systems, T.Higuchi and V. Stella, Vol. 14 of the A.C.S. Symposium Series, andBioreversible Carriers in Drug Design, Edward B. Roche, ea., AmericanPharmaceutical Association and Pergamon Press, 1987, each of which ishereby incorporated by reference herein.

Additional Pharmaceutical Components

The above-described pharmaceutical compositions including theacylsulfonamides may additionally include one or more pharmaceuticallyactive components. Suitable pharmaceutically active agents that may beincluded in the compositions include, for instance, anesthetics,antihypertensives, antianxiety agents, anticlotting agents,anticonvulsants, blood glucose-lowering agents, decongestants,antihistamines, antitussives, antineoplastics, beta blockers,anti-inflammatory agents, antipsychotic agents, cognitive enhancers,cholesterol-reducing agents, antiobesity agents, autoimmune disorderagents, anti-impotence agents, antibacterial and antifungal agents,hypnotic agents, anti-Parkinsonism agents, anti-Alzheimer's Diseaseagents, antibiotics, anti-depressants, and antiviral agents, amongothers.

The individual components of such combinations may be administeredeither sequentially or simultaneously in separate or combinedpharmaceutical formulations.

ABBREVIATIONS AND DEFINITIONS

The following definitions and methods are provided to better define thepresent disclosure and to guide those of ordinary skill in the art inthe practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

With regard to stereoisomers, it should be understood that a solid linedesignation for the bonds in the compositions corresponding to Formulae(1), (2), and (3) (and others herein) for attachment of an substituentgroup (e.g., Z₁, Z₂, and further substituents on these groups) to achiral carbon atom of the compound indicates that these groups may lieeither below or above the plane of the page (i.e.,

). All isomeric forms of the compounds disclosed herein arecontemplated, including racemates, racemic mixtures, and individualenantiomers or diastereomers.

The terms “acetal” and “ketal,” as used herein alone or as part ofanother group, denote the moieties represented by the followingformulae, respectively:

wherein X₁ and X₂ are independently hydrocarbyl, substitutedhydrocarbyl, heterocyclo, or heteroaryl, and X₃ is hydrocarbyl orsubstituted hydrocarbyl, as defined in connection with such terms, andthe wavy lines represent the attachment point of the acetal or ketalmoiety to another moiety or compound.

The term “acyl,” as used herein alone or as part of another group,denotes the moiety formed by removal of the hydroxyl group from thegroup —COOH of an organic carboxylic acid, e.g., X₄C(O)—, wherein X₄ isX¹, X¹O—, X¹X²N—, or X¹S—, X¹ is hydrocarbyl, heterosubstitutedhydrocarbyl, or heterocyclo, and X² is hydrogen, hydrocarbyl orsubstituted hydrocarbyl. Exemplary acyl moieties include acetyl,propionyl, benzoyl, pyridinylcarbonyl, and the like.

The term “acyloxy,” as used herein alone or as part of another group,denotes an acyl group as described above bonded through an oxygenlinkage (—O—), e.g., X₄C(O)O— wherein X₄ is as defined in connectionwith the term “acyl.”

The term “alkanol,” as used herein alone or as part of another group,denotes an alkyl radical having 1 to 10 carbon atoms, which issubstituted by one, two or three, or more, hydroxyl group(s). Examplesof alkanols include methanol, ethanol, n-propan-2-ol, n-propan-3-ol,isopropanol, i-butanol, and the like.

The term “alkanoyl,” as used herein, represents an alkyl group attachedto the parent molecular moiety through a carbonyl group. The alkanoylgroups of this invention can be optionally substituted with one or twogroups independently selected from the group consisting of hydroxyl andamino.

The term “alkanoylalkyl,” as used herein, represents an alkanoyl groupattached to the parent molecular moiety through an alkyl group.

The term “alkoxy,” as used herein alone or as part of another group,denotes an —OX₅ radical, wherein X₅ is as defined in connection with theterm “alkyl.” Exemplary alkoxy moieties include methoxy, ethoxy,propoxy, or 2-propoxy, n-, iso-, or tert-butoxy, and the like.

The term “alkenoxy,” as used herein alone or as part of another group,denotes an —OX₆ radical, wherein X₆ is as defined in connection with theterm “alkenyl.” Exemplary alkenoxy moieties include ethenoxy, propenoxy,butenoxy, hexenoxy, and the like.

The term “alkynoxy,” as used herein alone or as part of another group,denotes an —OX₇ radical, wherein X₇ is as defined in connection with theterm “alkynyl.” Exemplary alkynoxy moieties include ethynoxy, propynoxy,butynoxy, hexynoxy, and the like.

The term “alkoxyalkanoyl,” as used herein, represents an alkoxy groupattached to the parent molecular moiety through an alkanoyl group.

The term “alkoxyalkoxy,” as used herein, represents an alkoxy groupattached to the parent molecular moiety through another alkoxy group.

The term “alkoxyalkoxyalkyl,” as used herein, represents an alkoxyalkoxygroup attached to the parent molecular moiety through an alkyl group.

The term “alkoxyalkoxycarbonyl,” as used herein, represents analkoxyalkoxy group attached to the parent molecular moiety through acarbonyl group.

The term “alkoxyalkyl,” as used herein, represents an alkoxy groupattached to the parent molecular moiety through an alkyl group.

The term “alkoxycarbonyl,” as used herein, represents an alkoxy groupattached to the parent molecular moiety through a carbonyl group.

The term “alkoxycarbonylalkyl,” as used herein, represents analkoxycarbonyl group attached to the parent molecular moiety through analkyl group.

Unless otherwise indicated, the alkyl groups described herein arepreferably lower alkyl containing from one to eight carbon atoms in theprincipal chain and up to 20 carbon atoms. They may be straight orbranched chain or cyclic and include methyl, ethyl, propyl, isopropyl,butyl, hexyl and the like.

The term “alkylamino,” as used herein, represents —N(X₈)₂, wherein X₈ isalkyl.

The term “alkylaminoalkyl,” as used herein, represents an alkylaminogroup attached to the parent molecular moiety through an alkyl group.

The term “alkylaminocarbonyl,” as used herein, represents an alkylaminogroup attached to the parent molecular moiety through a carbonyl group.

The term “alkylaminocarbonylalkyl,” as used herein, represents analkylaminocarbonyl group attached to the parent molecular moiety throughan alkyl group.

The term “alkylidene,” as used herein, represents an alkyl groupattached to the parent molecular moiety through a carbon-carbon doublebond.

The term “alkylsulfanyl,” as used herein, represents an alkyl groupattached to the parent molecular moiety through a sulfur atom.

The term “alkylsulfanylalkyl,” as used herein, represents analkylsulfanyl group attached to the parent molecular moiety through analkyl group.

The term “alkylsulfonyl,” as used herein, represents an alkyl groupattached to the parent molecular moiety through a sulfonyl group.

The term “alkylsulfonylalkyl,” as used herein, represents analkylsulfonyl group attached to the parent molecular moiety through analkyl group.

The term “alkylene,” as used herein alone or as part of another group,denotes a linear saturated divalent hydrocarbon radical of one to eightcarbon atoms or a branched saturated divalent hydrocarbon radical ofthree to six carbon atoms unless otherwise stated. Exemplary alkylenemoieties include methylene, ethylene, propylene, 1-methylpropylene,2-methylpropylene, butylene, pentylene, and the like. Unless otherwiseindicated, one or more hydrogen atoms of the alkylene moieties can bereplaced and substituted with one or more of ═O, —OH, —OR_(Z), —COOH,—COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z),—SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo (including F, Cl, Brand I), among others, wherein each occurrence of R_(Z) may behydrocarbyl or substituted hydrocarbyl (e.g., substituted orunsubstituted alkyl, substituted or unsubstituted aryl, or substitutedor unsubstituted aralkyl.

Unless otherwise indicated, the alkenyl groups described herein arepreferably lower alkenyl containing from two to eight carbon atoms inthe principal chain and up to 20 carbon atoms. They may be straight orbranched chain or cyclic and include ethenyl, propenyl, isopropenyl,butenyl, isobutenyl, hexenyl, and the like.

Unless otherwise indicated, the alkynyl groups described herein arepreferably lower alkynyl containing from two to eight carbon atoms inthe principal chain and up to 20 carbon atoms. They may be straight orbranched chain and include ethynyl, propynyl, butynyl, isobutynyl,hexynyl, and the like.

Unless otherwise indicated, the terms “amine” or “amino,” as used hereinalone or as part of another group, represents a group of formula—N(X₉)(X₁₀), wherein X₉ and X₁₀ are independently hydrogen, hydrocarbyl,substituted hydrocarbyl, heteroaryl, or heterocyclo, or X₈ and X₉ takentogether form a substituted or unsubstituted alicyclic, aryl, orheterocyclic moiety, each as defined in connection with such term,typically having from 3 to 8 atoms in the ring. “Substituted amine,” forexample, refers to a group of formula —N(X₉)(X₁₀), wherein at least oneof X₉ and X₁₀ are other than hydrogen. “Unsubstituted amine,” forexample, refers to a group of formula —N(X₉)(X₁₀), wherein X₉ and X₁₀are both hydrogen.

By way of example, X₉ and X₁₀ may be independently selected fromhydrogen, alkanoyl, alkenyl, alkoxyalkyl, alkoxyalkoxyalkyl,alkoxycarbonyl, alkyl, alkylaminoalkyl, alkylaminocarbonylalkyl, aryl,arylalkyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkylcarbonyl,haloalkanoyl, haloalkyl, (heterocyclo)alkyl, heterocyclocarbonyl,hydroxyalkyl, an amino protecting group, —C(NH)NH₂, and —C(O)N(X₉)(X₁₀),wherein X₉ and X₁₀ are as previously defined; wherein the aryl; the arylpart of the arylalkyl; the cycloalkyl; the cycloalkyl part of the(cycloalkyl)alkyl and the cycloalkylcarbonyl; and the heterocycle partof the (heterocycle)alkyl and the heterocyclocarbonyl can be optionallysubstituted with one, two, three, four, or five substituentsindependently selected from the group consisting of alkanoyl, alkoxy,alkyl, cyano, halo, haloalkoxy, haloalkyl, hydroxyl, and nitro.

The term “aminoalkanoyl,” as used herein, represents an amino groupattached to the parent molecular moiety through an alkanoyl group.

The term “aminoalkyl,” as used herein, represents an amino groupattached to the parent molecular moiety through an alkyl group.

The term “aminocarbonyl,” as used herein, represents an amino groupattached to the parent molecular moiety through a carbonyl group.

The term “aminocarbonylalkyl,” as used herein, represents anaminocarbonyl group attached to the parent molecular moiety through analkyl group.

The term “aminosulfonyl,” as used herein, represents an amino groupattached to the parent molecular moiety through a sulfonyl group.

Unless otherwise indicated, the terms “amido” or “amide,” as used hereinalone or as part of another group, represents a group of formula—CON(X₉)(X₁₀), wherein X₉ and X₁₀ are as defined in connection with theterms “amine” or “amino.” In general, “amido” or “amide” groups may beeither substituted or unsubstituted. “Substituted amide,” for example,refers to a group of formula —CON(X₉)(X₁₀), wherein at least one of X₉and X₁₀ are other than hydrogen. “Unsubstituted amido,” for example,refers to a group of formula —CON(X₉)(X₁₀), wherein X₉ and X₁₀ are bothhydrogen.

The terms “amino protecting group,” “protected amino,” or “Pr” as usedherein denote moieties that block reaction at the protected amino groupwhile being easily removed under conditions that are sufficiently mildso as not to disturb other substituents of the various compounds. CommonN-protecting groups comprise benzyl and acyl groups such as acetyl,benzoyl, 2-bromoacetyl, 4-bromobenzoyl, tert-butylacetyl,carboxaldehyde, 2-chloroacetyl, 4-chlorobenzoyl, a-chlorobutyryl,4-nitrobenzoyl, o-nitrophenoxyacetyl, phthalyl, pivaloyl, propionyl,trichloroacetyl, and trifluoroacetyl; sulfonyl groups such asbenzenesulfonyl, and p-toluenesulfonyl; carbamate forming groups such asbenzyloxycarbonyl, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc),p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, allyloxycarbonyl,fluorenylmethoxycarbonyl (Fmoc), and the like. A variety of protectinggroups for the amino group and the synthesis thereof may be found in“Protective Groups in Organic Synthesis” by T. W. Greene and P. G. M.Wuts, John Wiley & Sons, 1999.

The terms “aryl” or “ar” as used herein alone or as part of anothergroup denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 12 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl or substituted naphthyl. For example, theterm “aryl,” may represent a phenyl group or a bicyclic or tricyclicfused ring system wherein one or more of the fused rings is a phenylgroup. Bicyclic fused ring systems are exemplified by a phenyl groupfused to a cycloalkyl group as defined herein, a cycloalkenyl group asdefined herein, or another phenyl group. Tricyclic fused ring systemsare exemplified by a bicyclic fused ring system fused to a cycloalkylgroup as defined herein, a cycloalkenyl group as defined herein, oranother phenyl group. Representative examples of aryl include, but arenot limited to, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl,naphthyl, phenyl, and tetrahydronaphthyl. Aryl groups having anunsaturated or partially saturated ring fused to an aromatic ring can beattached through the saturated or the unsaturated part of the group. Thearyl groups of this invention can be optionally substituted with one,two, three, four, or five substituents independently selected from thegroup consisting of alkanoyl, alkenyl, alkoxy, alkoxyalkanoyl,alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, alkyl, alkynyl, amino,aminoalkyl, aminocarbonyl, aminocarbonylalkyl, aminosulfonyl, aryl,aryloxy, arylsulfanyl, carbonyloxy, cyano, halo, haloalkoxy, haloalkyl,heterocycle, (heterocycle)alkyl, heterocyclecarbonylalkenyl,heterocyclecarbonylalkyl, hydroxy, hydroxyalkyl, nitro, oxo, and—C(NH)NH₂, wherein the aryl; the aryl part of the aryloxy and thearylsulfanyl; the heterocycle; and the heterocycle part of the(heterocycle)alkyl, the heterocyclecarbonylalkenyl, and theheterocyclecarbonylalkyl can be further optionally substituted with one,two, or three substituents independently selected from the groupconsisting of alkoxyalkanoyl, alkoxycarbonyl, alkyl, alkylsulfonyl,aminocarbonyl, aminosulfonyl, cyano, halo, haloalkoxy, haloalkyl,hydroxy, nitro, oxo, and —C(NH)NH₂. In addition, the heterocycle and theheterocycle part of the (heterocycle)alkyl, theheterocyclecarbonylalkenyl, and the heterocyclecarbonylalkyl can befurther optionally substituted with an additional aryl group, whereinthe aryl can be optionally substituted with one, two, or threesubstituents independently selected from the group consisting of alkoxy,alkyl, cyano, halo, hydroxy, and nitro.

The term “arylalkenyl,” as used herein, represents an aryl groupattached to the parent molecular moiety through an alkenyl group.

The term “arylalkoxy,” as used herein, represents an aryl group attachedto the parent molecular moiety through an alkoxy group.

The term “arylalkoxyalkanoyl,” as used herein, represents an arylalkoxygroup attached to the parent molecular moiety through an alkanoyl group.

The term “arylalkoxycarbonyl,” as used herein, represents an arylalkoxygroup attached to the parent molecular moiety through a carbonyl group.

The term “arylalkylsulfanyl,” as used herein, represents an arylalkylgroup attached to the parent molecular moiety through a sulfur atom.

The term “arylalkylsulfanylalkyl,” as used herein, represents anarylalkylsulfanyl group attached to the parent molecular moiety throughan alkyl group.

The term “arylalkylsulfonyl,” as used herein, represents an arylalkylgroup attached to the parent molecular moiety through a sulfonyl group.

The term “arylcarbonyl,” as used herein, represents an aryl groupattached to the parent molecular moiety through a carbonyl group.

The term “aryloxy,” as used herein, represents an aryl group attached tothe parent molecular moiety through an oxygen atom.

The term “aryloxyalkoxy,” as used herein, represents an aryloxy groupattached to the parent molecular moiety through an alkoxy group.

The term “aryloxyalkyl,” as used herein, represents an aryloxy groupattached to the parent molecular moiety through an alkyl group.

The term “arylsulfanyl,” as used herein, represents an aryl groupattached to the parent molecular moiety through a sulfur atom.

The term “arylsulfanylalkoxy,” as used herein, represents anarylsulfanyl group attached to the parent molecular moiety through analkoxy group.

The term “arylsulfanylalkyl,” as used herein, represents an arylsulfanylgroup attached to the parent molecular moiety through an alkyl group.The alkyl part of the arylsulfanylalkyl can be optionally substitutedwith one or two substituents independently selected from the groupconsisting of alkoxy, alkoxycarbonyl, amino, aminocarbonyl, arylalkoxy,azido, carboxy, cycloalkyl, halo, heterocycle, (heterocycle)alkoxy,(heterocycle)carbonyl, and hydroxy.

The term “arylsulfinyl,” as used herein, represents an aryl groupattached to the parent molecular moiety through a sulfinyl group.

The term “arylsulfinylalkyl,” as used herein, represents an arylsulfinylgroup attached to the parent molecular moiety through an alkyl group.The alkyl part of the arylsulfinylalkyl can be optionally substitutedwith one or two amino groups.

The term “arylsulfonyl,” as used herein, represents an aryl groupattached to the parent molecular moiety through a sulfonyl group.

The term “arylsulfonylalkyl,” as used herein, represents an arylsulfonylgroup attached to the parent molecular moiety through an alkyl group.The alkyl part of the arylsulfonylalkyl can be optionally substitutedwith one or two amino groups.

The term “arylene”, as used herein alone or part of another group refersto a divalent aryl radical of one to twelve carbon atoms. Non-limitingexamples of “arylene” include phenylene, pyridinylene, pyrimidinyleneand thiophenylene.

The terms “aralkyl,” “arylalkyl,” or “alkylene aryl,” as used hereinalone or as part of another group, denotes an -(alkylene)-X₁₁ radical,wherein X₁₁ is as defined in connection with the term “aryl.”Non-limiting examples of “aralkyl” or “alkylene aryl” moieties includebenzyl, —(CH₂)_(n)-phenyl where n is 2 to 6, or —CH-(phenyl)₂.

The terms “alkaryl” or “alkylaryl,” as used herein alone or as part ofanother group, denotes an -(arylene)-X₁₁ radical, wherein X₁₁ is asdefined in connection with the term “alkyl.”

The term “azido,” as used herein, represents a —N₃ moiety.

The term “carbocyclic,” as used herein alone or as part of anothergroup, denotes a ring wherein the atoms forming the ring backbone areselected from only carbon atoms. The carbocyclic rings may be optionallysubstituted, fully saturated or unsaturated, monocyclic or bicyclic,aromatic or nonaromatic, and generally include 3 to 20 carbon atoms.

The term “carbonyl,” as used herein, represents a —C(O)— moiety.

The term “carbonyloxy,” as used herein, represents an alkanoyl groupattached to the parent molecular moiety through an oxygen atom.

The term “carboxy,” as used herein, represents a —CO₂H moiety.

The term “carboxyalkyl,” as used herein, represents a carboxy groupattached to the parent molecular moiety through an alkyl group.

The term “cyano,” as used herein alone or as part of another group,denotes a group of formula —CN.

The term “cyanoalkyl,” as used herein, represents a cyano group attachedto the parent molecular moiety through an alkyl group.

The term “cycloalkyl,” as used herein alone or as part of another group,denotes a cyclic saturated monovalent bridged or non-bridged hydrocarbonradical of three to twelve carbon atoms. Exemplary cycloalkyl moietiesinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or adamantyl.By way of example, the term “cycloalkyl” may represent a saturated ringsystem having three to twelve carbon atoms and one to three rings.Examples of cycloalkyl groups include cyclopropyl, cyclopentyl,bicyclo(3.1.1)heptyl, adamantyl, and the like. The cycloalkyl groups ofthis invention can be optionally substituted with one, two, three, four,or five substituents independently selected from the group consisting ofalkoxy, alkoxycarbonyl, alkyl, aminoalkyl, arylalkoxy, aryloxy,arylsulfanyl, halo, haloalkoxy, haloalkyl, and hydroxy, wherein the arylpart of the arylalkoxy, the aryloxy, and the arylsulfanyl can be furtheroptionally substituted with one, two, or three substituentsindependently selected from the group consisting of alkoxy, alkyl, halo,haloalkoxy, haloalkyl, and hydroxy.

The term “cycloalkylalkoxy,” as used herein, represents a cycloalkylgroup attached to the parent molecular moiety through an alkoxy group.

The term “(cycloalkyl)alkyl,” as used herein, represents a cycloalkylgroup attached to the parent molecular moiety through an alkyl group.

The term “cycloalkylcarbonyl,” as used herein, represents a cycloalkylgroup attached to the parent molecular moiety through a carbonyl group.

The term “cycloalkyloxy,” as used herein, represents a cycloalkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “cycloalkenyl,” as used herein, represents a non-aromatic ringsystem having three to ten carbon atoms and one to three rings, whereineach five-membered ring has one double bond, each six-membered ring hasone or two double bonds, each seven- and eight-membered ring has one tothree double bonds, and each nine- to ten-membered ring has one to fourdouble bonds. Examples of cycloalkenyl groups include cyclohexenyl,octahydronaphthalenyl, norbornylenyl, and the like. The cycloalkenylgroups of this invention can be optionally substituted with one, two,three, four, or five substituents independently selected from the groupconsisting of alkoxy, alkoxycarbonyl, alkyl, aminoalkyl, arylalkoxy,aryloxy, arylsulfanyl, halo, haloalkoxy, haloalkyl, and hydroxy, whereinthe aryl part of the arylalkoxy, the aryloxy, and the arylsulfanyl canbe further optionally substituted with one, two, or three substituentsindependently selected from the group consisting of alkoxy, alkyl, halo,haloalkoxy, haloalkyl, and hydroxy.

The term “cycloalkenylalkyl,” as used herein, represents a cycloalkenylgroup attached to the parent molecular moiety through an alkyl group.

The term “ester,” as used herein alone or as part of another group,denotes a group of formula —COOX₁₂ wherein X₁₂ is alkyl or aryl, each asdefined in connection with such term.

The term “ether,” as used herein alone or as part of another group,includes compounds or moieties which contain an oxygen atom bonded totwo carbon atoms. For example, ether includes “alkoxyalkyl” which refersto an alkyl, alkenyl, or alkynyl group substituted with an alkoxy group.

The term “formyl,” as used herein, represents a —CHO moiety.

The term “formylalkyl,” as used herein, represents a formyl groupattached to the parent molecular moiety through an alkyl group.

The terms “halide,” “halogen” or “halo” as used herein alone or as partof another group refer to chlorine, bromine, fluorine, and iodine.

The term “haloalkyl,” as used herein, represents an alkyl groupsubstituted by one, two, three, or four halogen atoms.

The term “haloalkanoyl,” as used herein, represents a haloalkyl groupattached to the parent molecular moiety through a carbonyl group.

The term “haloalkoxy,” as used herein, represents a haloalkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “heteroatom” shall mean atoms other than carbon and hydrogen.

The terms “heteroaralkyl” and “alkylene heteroaryl,” as used hereinalone or as part of another group, denotes an -(alkylene)-X₁₃ radical,wherein X₁₃ is as defined in connection with the term “heteroaryl.”Non-limiting examples of “heteroaralkyl” or “alkylene heteroaryl”moieties include —(CH₂)_(n)-indolyl where n is 1 to 6.

The term “heteroalkylene,” as used herein, represents a divalent groupof two to eight atoms derived from a saturated straight or branchedchain containing one or two heteroatoms independently selected from thegroup consisting of nitrogen, oxygen, and sulfur, wherein the remainingatoms are carbon. The heteroalkylene groups of the present invention canbe attached to the parent molecular moiety through the carbon atoms orthe heteroatoms in the chain.

The term “heteroalkenylene,” as used herein, represents a divalent groupof three to eight atoms derived from a straight or branched chaincontaining at least one carbon-carbon double bond that contains one ortwo heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur, wherein the remaining atoms are carbon.The heteroalkenylene groups of the present invention can be attached tothe parent molecular moiety through the carbon atoms or the heteroatomsin the chain.

The term “heterocyclo” or “heterocycle,” as used herein, represents amonocyclic, bicyclic, or tricyclic ring system wherein one or more ringsis a four-, five-, six-, or seven-membered ring containing one, two, orthree heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. Monocyclic ring systems are exemplified byany 3- or 4-membered ring containing a heteroatom independently selectedfrom the group consisting of oxygen, nitrogen and sulfur; or a 5-, 6- or7-membered ring containing one, two or three heteroatoms wherein theheteroatoms are independently selected from the group consisting ofnitrogen, oxygen and sulfur. The 3- and 4-membered rings have no doublebonds, the 5-membered ring has from 0-2 double bonds and the 6- and7-membered rings have from 0-3 double bonds. Representative examples ofmonocyclic ring systems include, but are not limited to, azetidine,azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan,imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline,isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine,oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline,oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole,pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole,pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine,tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole,thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholinesulfone, thiopyran, triazine, triazole, trithiane, and the like.Bicyclic ring systems are exemplified by any of the above monocyclicring systems fused to an aryl group as defined herein, a cycloalkylgroup as defined herein, a cycloalkenyl group, as defined herein, oranother monocyclic heterocycle ring system. Representative examples ofbicyclic ring system include but are not limited to, benzimidazole,benzothiazole, benzothiophene, benzoxazole, benzofuran, benzopyran,benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole,indole, indoline, indolizine, naphthyridine, isobenzofuran,isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine,pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline,tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and thelike. Tricyclic rings systems are exemplified by any of the abovebicyclic ring systems fused to an aryl group as defined herein, acycloalkyl group as defined herein, a cycloalkenyl group as definedherein, or another monocyclic heterocycle ring system. Representativeexamples of tricyclic ring systems include, but are not limited to,acridine, carbazole, carboline, dibenzofuran, dibenzothiophene,naphthofuran, naphthothiophene, oxanthrene, phenazine, phenoxathiin,phenoxazine, phenothiazine, thianthrene, thioxanthene, xanthene, and thelike. Heterocycle groups can be attached to the parent molecular moietythrough a carbon atom or a nitrogen atom in the group.

The heterocyclo groups of the present invention can be optionallysubstituted with one, two, three, four, or five substituentsindependently selected from the group consisting of alkanoyl,alkanoylalkyl, alkenyl, alkoxy, alkoxyalkoxycarbonyl, alkoxyalkyl,alkoxycarbonyl, alkoxycarbonylalkyl, alkyl, alkylsulfanylalkyl, alkynyl,amino, aminoalkanoyl, aminoalkyl, aminocarbonyl, aminocarbonylalkyl,aminosulfonyl, aryl, arylalkoxyalkanoyl, arylalkoxycarbonyl, arylalkyl,arylalkylsulfonyl, arylcarbonyl, aryloxy, arylsulfanyl,arylsulfanylalkyl, arylsulfonyl, carbonyloxy, carboxy, cyano,cyanoalkyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkylcarbonyl, formyl,formylalkyl, halo, haloalkoxy, haloalkyl, heterocycle,(heterocycle)alkyl, (heterocycle)alkylidene, heterocyclecarbonyl,heterocyclecarbonylalkyl, hydroxy, hydroxyalkyl, nitro, oxo, spirocycle,spiroheterocycle, and —C(NH)NH₂; wherein the aryl; the aryl part of thearylalkylsulfonyl, the arylcarbonyl, the aryloxy, thearylalkoxyalkanoyl, the arylalkoxycarbonyl, the arylalkyl, thearylsulfanyl, the arylsulfanylalkyl, and the arylsulfonyl; theheterocycle; and the heterocycle part of the (heterocycle)alkyl, the(heterocycle)alkylidene, the heterocyclecarbonyl, and theheterocyclecarbonylalkyl can be further optionally substituted with one,two, three, four, or five substituents independently selected from thegroup consisting of alkanoyl, alkoxy, alkoxyalkoxycarbonyl,alkoxycarbonyl, alkyl, halo, haloalkoxy, haloalkyl, hydroxy,hydroxyalkyl, and nitro.

The term “(heterocyclo)alkoxy,” as used herein, represents a heterocyclogroup attached to the parent molecular moiety through an alkoxy group.

The term “(heterocyclo)alkyl,” as used herein, represents a heterocyclogroup attached to the parent molecular moiety through an alkyl group.

The term “(heterocyclo)alkylidene,” as used herein, represents aheterocyclo group attached to the parent molecular moiety through analkylidene group.

The term “heterocyclocarbonyl,” as used herein, represents a heterocyclogroup attached to the parent molecular moiety through a carbonyl group.

The term “heterocyclocarbonylalkenyl,” as used herein, represents aheterocyclecarbonyl group attached to the parent molecular moietythrough an alkenyl group.

The term “heterocyclocarbonylalkyl,” as used herein, represents aheterocyclocarbonyl group attached to the parent molecular moietythrough an alkyl group.

The term “(heterocyclo)oxy,” as used herein, represents a heterocyclogroup attached to the parent molecular moiety through an oxygen atom.

The term “(heterocyclo)sulfanyl,” as used herein, represents aheterocyclo group attached to the parent molecular moiety through asulfur atom.

The term “(heterocyclo)sulfanylalkyl,” as used herein, represents aheterocyclosulfanyl group attached to the parent molecular moietythrough an alkyl group.

The term “heteroaromatic” or “heteroaryl” as used herein alone or aspart of another group denote optionally substituted aromatic groupshaving at least one heteroatom in at least one ring, and preferably 5 or6 atoms in each ring. The heteroaromatic group preferably has 1 or 2oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in thering, and may be bonded to the remainder of the molecule through acarbon or heteroatom. Exemplary heteroaromatics include furyl, thienyl,pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl, or isoquinolinyl andthe like. Exemplary substituents include one or more of the followinggroups: hydrocarbyl, substituted hydrocarbyl, keto, hydroxyl, protectedhydroxyl, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen,amido, amino, nitro, cyano, thiol, ketals, acetals, esters and ethers.

The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwiseindicated, these moieties preferably comprise 1 to 20 carbon atoms.

The term “hydroxy” or “hydroxyl,” as used herein alone or as part ofanother group, denotes a group of formula —OH.

The term “hydroxyalkyl,” as used herein, represents a hydroxy groupattached to the parent molecular moiety through an alkyl group.

The term “hydroxyl protecting group,” as used herein alone or as part ofanother group, denote a group capable of protecting a free hydroxylgroup (“protected hydroxyl”) which, subsequent to the reaction for whichprotection is employed, may be removed without disturbing the remainderof the molecule. Exemplary hydroxylprotecting groups include ethers(e.g., allyl, triphenylmethyl (trityl or Tr), benzyl, p-methoxybenzyl(PMB), p-methoxyphenyl (PMP)), acetals (e.g., methoxymethyl (MOM),β-methoxyethoxymethyl (MEM), tetrahydropyranyl (THP), ethoxy ethyl (EE),methylthiomethyl (MTM), 2-methoxy-2-propyl (MOP),2-trimethylsilylethoxymethyl (SEM)), esters (e.g., benzoate (Bz), allylcarbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethylcarbonate), silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl(TES), triisopropylsilyl (TIPS), triphenylsilyl (TPS),t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS) and the like.A variety of protecting groups for the hydroxyl group and the synthesisthereof may be found in “Protective Groups in Organic Synthesis” by T.W. Greene and P. G. M. Wuts, John Wiley & Sons, 1999.

The term “keto,” as used herein alone or as part of another group,denotes a double bonded oxygen moiety (i.e., ═O).

The term “nitro,” as used herein alone or as part of another group,denotes a group of formula —NO₂.

The term “oxo,” as used herein, represents a (═O) moiety.

The term “spirocycle,” as used herein, represents an alkyl diradical oftwo to eight atoms, each end of which is attached to the same carbonatom of the parent molecular moiety.

The term “spiroheterocycle,” as used herein, represents a heteroalkylenediradical, each end of which is attached to the same carbon atom of theparent molecular moiety. Examples of spiroheterocycles includedioxolanyl, tetrahydrofuranyl, pyrrolidinyl, and the like.

The term “sulfinyl,” as used herein, represents a —S(═O)— moiety.

The term “sulfonyl,” as used herein, represents —S(═O)₂— moiety

Unless otherwise indicated, the “substituted hydrocarbyl” moietiesdescribed herein are hydrocarbyl moieties which are substituted with atleast one atom other than carbon, including moieties in which a carbonchain atom is substituted with a hetero atom such as nitrogen, oxygen,silicon, phosphorous, boron, sulfur, or a halogen atom. Thesesubstituents include halogen, heterocyclo, alkoxy, alkenoxy, alkynoxy,aryloxy, hydroxyl, protected hydroxyl, keto, acyl, acyloxy, nitro,amino, amido, nitro, cyano, thiol, ketals, acetals, esters, ethers, andthioethers.

The term “thioester,” as used herein alone or as part of another group,denotes a group of formula —C(O)—S—X₁₄, wherein X₁₄ is alkyl or aryl asdefined in connection with such term.

The term “thioether,” as used herein alone or as part of another group,denotes compounds and moieties that contain a sulfur atom bonded to twodifferent carbon or hetero atoms (i.e., —S—), and also includescompounds and moieties containing two sulfur atoms bonded to each other,each of which is also bonded to a carbon or hetero atom (i.e.,dithioethers (—S—S—)). Examples of thioethers include, but are notlimited to, alkylthioalkyls, alkylthioalkenyls, and alkylthioalkynyls.The term “alkylthioalkyls” includes compounds with an alkyl, alkenyl, oralkynyl group bonded to a sulfur atom that is bonded to an alkyl group.Similarly, the term “alkylthioalkenyls” and alkylthioalkynyls” refer tocompounds or moieties where an alkyl, alkenyl, or alkynyl group isbonded to a sulfur atom that is covalently bonded to an alkynyl group.

The term “thiol,” as used herein alone or as part of another group,denotes a group of formula —SH.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing the scope ofthe invention defined in the appended claims. Furthermore, it should beappreciated that all examples in the present disclosure are provided asnon-limiting examples.

In general, all reactions were run under an atmosphere of nitrogenunless otherwise indicated. Prior to use of solvents in reactions, theywere purified by passing the degassed solvents through a column ofactivated alumina and transferred by an oven-dried syringe or cannula.Thin layer chromatography was performed on Merck TLC plates (silica gel60 F₂₅₄). ¹H-NMR and ¹³C-NMR were recorded on a Varian Inova 400 (400MHz) or a Bruker Avance DPX-250 (250 MHz) instrument. The HRMS data weremeasured on an Agilent 1100 Series MSD/TOF with electrospray ionization.LC/MS data were measured on an Agilent 1100 LC/MSD-VL with electrosprayionization. Sulfonyl azide (SZ8) prepared as reported procedure.

Example 1 Preparation of Building Blocks

1.1 Sulfonylazide (SZ8)

A saturated solution of sodium azide (1.2 g, 18.5 mmol) in water wasadded slowly to a saturated solution of 1 (5 g, 18.5 mmol) in acetone atroom temperature. The mixture was stirred at room temperature for 3hours. Ethyl acetate (50 mL) and saturated aqueous potassium carbonatesolution (50 mL) were added. After extraction with ethyl acetate (50mL×3), the combined organic phases were dried over anhydrous sodiumsulfate and concentrated. The product 2 (SZ8) (4.0 g, 78%) was obtainedby flash chromatography (hexanes:EtOAc=24:1). Rf=0.4(hexanes:EtOAc=8:1). ¹H-NMR (400 MHz, CDCl₃) δ: 7.91 (d, J=8.3 Hz, 2H),7.60 (d, J=8.3 Hz, 2H), 4.50 (s, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ:145.2, 138.4, 130.4, 128.2, 31.1 ppm. HRMS (ESI⁺) for [M+NH₄]⁺;calculated: 292.97024, found: 292.96949 (error m/z=−2.54 ppm).

A mixture of (SZ8) (100 mg, 0.36 mmol), 3 (60 mg, 0.36 mmol) andpotassium carbonate (100 mg, 0.72 mmol) in acetonitrile and water (9:1)was stirred at room temperature for 12 hours. The reaction mixture wasthen mixed with ethyl acetate (20 mL) and water (20 mL), and extractedwith ethyl acetate (20 mL×3). The combined organic phases were driedover anhydrous sodium sulfate and concentrated. Product (SZ1) (110 mg,85%) was obtained by flash chromatography (hexane:EtOAc=12:1). Rf=0.45(hexanes: EtOAc=4:1). ¹H-NMR (400 MHz, CDCl₃) δ: 7.91 (d, J=8.2 Hz, 2H),7.62 (d, J=8.2 Hz, 2H), 7.26-7.24 (m, 2H), 6.94-6.84 (m, 3H), 3.66 (s,2H), 3.23-3.20 (m, 4H), 2.64-2.61 (m, 4H) ppm. ¹³C-NMR (100 MHz, CDCl₃)δ: 151.2, 146.5, 137.1, 129.9, 129.1, 127.5, 119.8, 116.1, 62.2, 53.2,49.1 ppm. HRMS (ESI⁺) for [M+H]⁺; calculated: 358.13322, found:358.13320 (error m/z=−0.07 ppm).

1.2 Sulfonylazide (SZ2)

The mixture of (SZ8) (100 mg, 0.36 mmol), 4 (50 mg, 0.36 mmol) andpotassium carbonate (100 mg, 0.72 mmol) in acetonitrile and water (9:1),was stirred at room temperature for 12 hours. After mixed with ethylacetate (20 mL) and water (20 mL), the system was extracted by ethylacetate (20 mL×3). The combined organic phase was dried by anhydroussodium sulfate and concentrated. Product (SZ2) (100 mg, 83%) wasobtained by flash chromatography (hexane:EtOAc=14:1; Rf=0.5 inhexane:EtOAc=4:1). ¹H-NMR (400 MHz, CDCl₃) δ: 7.84 (d, J=8.2 Hz, 2H),7.48 (d, J=8.1 Hz, 2H), 7.30-7.16 (m, 5H), 3.63 (s, 2H), 2.82 (t, J=8.0Hz, 2H), 2.66 (t, J=8.0 Hz, 2H), 2.30 (s, 3H) ppm. ¹³C-NMR (100 MHz,CDCl₃) δ: 147.7, 140.2, 136.7, 129.7, 128.7, 128.3, 127.4, 126.1, 61.6,59.2, 42.2, 33.9 ppm. HRMS (ESI⁺) for [M+H]⁺; calculated: 331.12232,found: 331.12269 (error m/z=1.11 ppm).

1.3 Sulfonylazide (SZ3)

(SZ3) was prepared starting from 5 using the procedure described for thepreparation of 2 with 60% yield (hexane:EtOAc=2:1; Rf=0.25 inhexane:EtOAc=1:1). ¹H-NMR (400 MHz, CDCl₃) δ: 8.10 (s, 1H), 7.84 (d,J=8.2 Hz, 2H), 7.74 (d, J=8.2 Hz, 2H), 2.21 (s, 3H) ppm. ¹³C-NMR (100MHz, CDCl₃) δ: 169.0, 143.8, 132.2, 128.7, 119.4, 24.5 ppm. HRMS (ESI⁺)for [M+NH₄]⁺; calculated: 258.06554, found: 258.06476 (error m/z=−3.02ppm).

1.4 Sulfonylazide (SZ4)

A saturated solution of sodium azide (280 mg, 4.30 mmol) in water wasadded slowly to a saturated solution of 6 (see Wendt et al., J. Med.Chem. 2006, 49, 1165-1181) (500 mg, 2.1 mmol) in acetone at 0° C. Themixture was stirred at 0° C. for 3 hours. Ethyl acetate (20 mL) andsaturated aqueous potassium carbonate solution (20 mL) were added to themixture and after extraction with ethyl acetate (20 mL×3), the combinedorganic phases were dried over anhydrous sodium sulfate andconcentrated. Product 7 was used without further purification in thenext reaction.

A mixture of compounds 7 (see Wendt et al., supra) (280 mg, more than0.91 mmol) and 8 (224 mg, 0.91 mmol) in triethylamine (5 mL) was stirredovernight at room temperature. To the reaction mixture was added silica(600 mg) and the solvent was removed under reduced pressure. Product(SZ4) (260 mg, 68.5% over two steps) was obtained by flashchromatography (hexane:EtOAc=8:1-2:1; Rf=0.4 in hexane:EtOAc=2:1).¹H-NMR (400 MHz, CDCl₃) δ: 8.77 (d, J=2.4 Hz, 1H), 8.71 (d, J=2.4 Hz,1H), 7.79 (dd, J=9.2, 2.4 Hz, 1H), 7.39 (dd, J=8.4, 1.2 Hz, 2H),7.30-7.24 (m, 3H), 6.85 (d, J=9.2 Hz, 1H), 3.60 (dd, J=9.0, 6.4 Hz, 2H),3.22 (t, J=6.8 Hz, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 148.1, 134.2,133.6, 131.3, 129.8, 129.5, 128.5, 127.7, 124.5, 115.0, 42.3, 33.3 ppm.HRMS (ESI⁺) for [M+H]⁺; calculated: 380.04817, found: 380.04795 (errorm/z=−0.59 ppm).

1.5 Sulfonylazide (SZ5)

The known (SZ5) (Waser et al., J. Am. Chem. Soc. 2006, 128(35),11693-11712) was prepared starting from 9 using the procedure describedfor the preparation of 2 in 87% yield. (DCM: MeOH=60:1; Rf=0.4 in DCM:MeOH=20:1) ¹H-NMR (250 MHz, CDCl₃) δ: 7.84 (d, J=8.4 Hz, 2H), 7.41 (d,J=8.1 Hz, 2H), 2.48 (s, 3H) ppm.

1.6 Sulfonylazide (SZ6)

The mixture of (SZ8) (100 mg, 0.36 mmol), 10 (55 mg, 0.36 mmol) andpotassium carbonate (100 mg, 0.72 mmol) in acetonitrile and water (9:1),was stirred at room temperature for 12 hours. The reaction mixture wasthen mixed with ethyl acetate (20 mL) and water (20 mL), and extractedby ethyl acetate (20 mL×3). The combined organic phases were dried overanhydrous sodium sulfate and concentrated. Product (SZ6) (89 mg, 71%)was obtained by flash chromatography (hexane:EtOAc=3:1). Rf=0.45(hexanes:EtOAc=1:1). ¹H-NMR (400 MHz, CDCl₃) δ: 7.86 (d, J=8.4 Hz, 2H),7.53 (d, J=8.4 Hz, 2H), 7.33-7.18 (m, 5H), 3.88 (s, 3H), 3.08 (t, J=6.0Hz, 2H), 2.83 (t, J=6.0 Hz, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 148.2,136.8, 135.5, 129.8, 129.0, 127.6, 126.4, 52.6, 47.5, 34.2 ppm. HRMS(ESI⁺) for [M+H]⁺; calculated: 349.07874, found: 349.07937 (errorm/z=1.79 ppm).

1.7 Sulfonyl Azide (SZ7)

The mixture of compound 1 (664 mg, 2 mmol), SOCl₂ (4 mL), and DMF (0.1mL) were refluxed for 2 h. Cold water (15 mL) was added, the mixture wasextracted with DCM (4×15 mL), and the combined organic extracts weredried over Na₂SO₄. A quick filtration through a pad of silica gel,evaporation, and vacuum-drying gave the crude product 2 according tosimilar procedure (see Paruch et al., J. Org. Chem.; 2000; 65,8774-8782). And compound 2 was used directly for next step.

A solution of compounds 2 and 3 (155 mg, 1 mmol) and potassium carbonate(200 mg, 1.44 mmol) in CHCl₃, was stirred at room temperature for 12hours. The reaction mixture was then concentrated, mixed with ethylacetate (20 mL) and water (20 mL), and extracted by ethyl acetate (20mL×3). The combined organic phases were dried over anhydrous sodiumsulfate and concentrated. The obtained crude product 4 was dissolved inacetone and added a solution of sodium azide (70 mg, 1 mmol) in waterdropwise at 0° C. The mixture was stirred at 0° C. for 3 hours. Ethylacetate (20 mL) and saturated aqueous potassium carbonate solution (20mL) were added to the mixture and after extraction with ethyl acetate(20 mL×3), the combined organic phases were dried over anhydrous sodiumsulfate and concentrated. Product (SZ7) (315 mg, 70%) was obtained byflash chromatography (hexane:EtOAc=4:1; Rf=0.6 in hexane:EtOAc=1:1).

1.8 Sulfonyl Azide (SZ9)

The mixture of 5 (276 mg, 1 mmol), (SZ8) (77 mg, 0.5 mmol) and potassiumcarbonate (200 mg, 1.44 mmol) in acetonitrile and water (9:1), wasstirred at room temperature for 12 hours. After mixed with ethyl acetate(20 mL) and water (20 mL), the system was extracted by ethyl acetate (20mL×3). The combined organic phase was dried by anhydrous sodium sulfateand concentrated. Product (SZ9) (154 mg, 60%) was obtained by flashchromatography (hexane:EtOAc=6:1; Rf=0.2 in hexane:EtOAc=4:1). ¹H-NMR(400 MHz, CDCl₃) δ: 7.87 (d, J=8.3 Hz, 4H), 7.57 (d, J=8.3 Hz, 4H),7.24-7.15 (m, 5H), 3.72 (s, 4H), 3.07 (t, J=7.2 Hz, 2H), 2.76 (t, J=7.1Hz, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 146.6, 137.4, 129.6, 129.2,129.0, 127.9, 127.6, 126.3, 57.9, 52.7, 31.5 ppm. HRMS (ESI⁺) for[M+H]⁺; calculated: 544.08899, found: 544.08874 (error m/z=−0.46 ppm).

1.9 Sulfonyl Azide (SZ10)

The solution of commercially available compound 6 (1 g, 3.74 mmol) inDCM was bubbled by ammonia gas at 0° C. for 10 min. After mixed with DCM(20 mL) and water (20 mL), the system was extracted by DCM (20 mL×3).The combined organic phase was dried by anhydrous sodium sulfate andconcentrated. Product 7 (900 mg, 96%) was obtained by flashchromatography (hexane:EtOAc=3:1; Rf=0.5 in hexane:EtOAc=1:1). ¹H-NMR(400 MHz, DMSO-d6) δ: 7.81 (d, J=8.4 Hz, 2H), 7.63 (d, J=8.4 Hz, 2H),7.39 (s, 2H), 4.76 (s, 2H) ppm. ¹³C-NMR (100 MHz, DMSO-d6) δ: 143.8,141.9, 129.8, 126.0, 32.9 ppm.

The compound 7 (900 mg, 3.6 mmol), benzylaldehyde (381 mg, 3.6 mmol) andpara-methylbenzylsulfonic acid (10 mg) in benzene had been refluxed for10 h with Dean-stack condenser. The system was cooled down and extractedby ethyl acetate (20 mL×3). The combined organic phase was dried byanhydrous sodium sulfate, concentrated, and gave the product 8, whichwas used for next step directly. The mixture of 8, the known (SZ6) (1.25g, 3.6 mmol) and potassium carbonate (1.0 g, 7.2 mmol) in acetonitrileand water (9:1), was refluxing for 24 hours. After cooled down and mixedwith ethyl acetate (20 mL) and water (20 mL), the system was extractedby ethyl acetate (20 mL×3). The combined organic phase was dried byanhydrous sodium sulfate and concentrated. The interesting thing here isthe hydrolyzation of the imine happened smoothly under this basiccondition. And product (SZ10) (930 mg, 50%) was obtained by flashchromatography (hexane:EtOAc=2:1; Rf=0.2 in hexane:EtOAc=2:1). ¹H-NMR(400 MHz, CDCl₃) δ: 7.83 (d, J=8.4 Hz, 4H), 7.55 (d, J=8.4 Hz, 2H), 7.47(d, J=8.4 Hz, 2H), 7.24-7.14 (m, 5H), 5.00 (bs, 2H), 3.68 (s, 2H), 3.67(s, 2H), 3.06 (t, J=6.8 Hz, 2H), 2.75 (t, J=6.8 Hz, 2H) ppm. ¹³C-NMR(100 MHz, CDCl₃) δ: 147.0, 144.1, 140.9, 137.1, 135.8, 129.6, 129.2,129.1, 128.9, 127.5, 126.5, 126.2, 58.1, 58.0, 53.0, 31.6 ppm. HRMS(ESI⁺) for [M+H]⁺; calculated: 518.09849, found: 518.09993 (errorm/z=2.76 ppm).

1.10 Sulfonyl Azide (SZ11)

(SZ11) was prepared through the procedure described for the preparationof (SZ9) in 87% yield by using (SZ8) and known compound 9, which isprepared according to the reported method. ¹H-NMR (400 MHz, CDCl₃) δ:7.82 (d, J=8.4 Hz, 2H), 7.53 (d, J=8.0 Hz, 2H), 7.36-7.22 (m, 5H), 6.62(s, 1H), 6.20 (s, 1H), 4.57 (s, 1H), 3.83 (d, J=14.8 Hz, 1H), 3.80 (s,3H), 3.56 (s, 3H), 3.39 (d, J=14.8 Hz, 1H), 3.00-2.98 (m, 2H), 2.71 (d,J=15.2 Hz, 1H), 2.52 (d, J=15.2 Hz, 1H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ:147.8, 147.2, 146.8, 143.4, 136.3, 129.5, 129.2, 129.1, 128.1, 127.2,127.0, 126.2, 111.4, 110.6, 68.1, 57.9, 55.4, 47.3, 28.2 ppm. HRMS(ESI⁺) for [M+H]⁺; calculated: 465.15965, found: 465.15970 (errorm/z=0.1 ppm).

1.11 Sulfonyl Azide (SZ12)

(SZ12) was prepared starting from 10 and (SZ8) using the proceduredescribed for the preparation of (SZ9) in 67% yield. ¹H-NMR (400 MHz,CDCl₃) δ: 7.90 (d, J=6.8 Hz, 2H), 7.59-7.25 (m, 5H), 6.84 (d, J=7.6 Hz,1H), 4.53 (s, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 148.0, 146.2, 130.0,128.3, 128.1, 128.0, 127.6, 118.7, 112.8, 106.7, 47.3 ppm. HRMS (ESI⁺)for [M+K]⁺; calculated: 372.01633, found: 372.01449 (error m/z=−4.94ppm).

1.12 Sulfonyl Azide (SZ13)

(SZ13) was prepared starting from 11 and (SZ8) using the proceduredescribed for the preparation of (SZ9) in 40% yield. ¹H-NMR (400 MHz,CDCl₃) δ: 7.89-7.84 (m, 3H), 7.61-7.33 (m, 5H), 4.48 (s, 2H) ppm.¹³C-NMR (100 MHz, CDCl₃) δ: 143.2, 141.1, 134.3, 134.0, 129.0, 128.5,127.7, 125.8, 125.1, 53.6 ppm. HRMS (ESI⁺) for [M+NH₄]⁺; calculated:352.0716, found: 352.0719 (error m/z=0.8 ppm).

1.13 Sulfonyl Azide (SZ14)

(SZ14) was prepared starting from 12 and (SZ8) using the proceduredescribed for the preparation of (SZ9) in 45% yield. ¹H-NMR (400 MHz,CDCl₃) δ: 8.08 (dd, J=8.8, 3.6 Hz, 1H), 7.87-7.82 (m, 4H), 7.50-7.48 (m,5H), 7.36 (d, J=2.0 Hz, 1H), 6.91 (dd, J=8.8, 2.0 Hz, 1H), 6.75 (d,J=4.0 Hz, 1H) 4.47 (s, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 177.1,163.8, 156.3, 152.7, 142.8, 134.4, 131.8, 131.0, 129.0, 128.9, 128.6,127.4, 126.2, 122.6, 119.4, 112.0, 107.6, 53.6 ppm. HRMS (ESI⁺) for[M+H]⁺; calculated: 434.08052, found: 434.07955 (error m/z=−2.24 ppm).

1.14 Sulfonyl Azide (SZ15)

(SZ15) was prepared starting from 13 and known (SZ6) using the proceduredescribed for the preparation of (SZ9) in 54% yield. ¹H-NMR (400 MHz,CDCl₃) δ: 7.78 (d, J=8.0 Hz, 2H), 7.51 (d, J=8.0 Hz, 2H), 7.18-7.11 (m,7H), 6.94-6.90 (m, 1H), 3.85 (s, 2H), 3.73 (s, 2H), 3.05 (t, J=7.6 Hz,2H), 2.77 (t, J=7.6 Hz, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 161.9 (d,¹J_(CF)=247 Hz), 147.8, 136.6, 136.3, 135.8, 129.5, 129.4, 128.9 (d,²J_(CF)=26 Hz), 127.1, 126.0, 125.5, 124.1 (d, ³J_(CF)=16.8 Hz), 113.9(d, ²J_(CF)=23.3 Hz), 57.8, 53.6, 49.4, 31.1 ppm.

1.15 Sulfonyl Azide (SZ16)

(SZ16) was prepared by two steps. First, the mixture of 14 (570 mg, 3.3mmol) and mesyl chloride (0.45 ml, 5.2 mmol) inN,N-Diisopropylethylamine (10 ml) was stirred for 3 hours and then (SZ6)(1.15 g, 3.3 mmol) was added. After stirring for another 3 hours, 30 mlethyl acetate and 30 ml CuSO₄ aqueous solution were added. The aqueousphase was extracted twice with 30 ml ethyl acetate and the combinedorganic phase is dried over Na₂SO₄ and concentrated down. (SZ16) (310mg, 19% over two steps) was obtained by flash chromatography(hexane:EtOAc=6:1; Rf=0.3 in hexane:EtOAc=2:1). ¹H-NMR (400 MHz, CDCl₃)δ: 8.01 (d, J=6.0 Hz, 1H), C 7.58-7.56 (m, 3H), 7.23-7.14 (m, 6H), 3.70(s, 2H), 3.63 (s, 2H), 3.05 (t, J=7.3 Hz, 2H), 2.74 (t, J=7.6 Hz, 2H)ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 154.6 (¹J_(CF)=263.5 Hz), 146.6, 137.3,135.7 (²J_(CF)=24.4 Hz), 135.3 (³J_(CF)=8.4 Hz), 129.6, 129.2, 128.9,127.6, 126.3, 125.8, 118.4 (²J_(CF)=20.6 Hz) ppm. HRMS (ESI⁺) for[M+H]⁺; calculated: 502.10135, found: 502.09963 (error m/z=−3.42 ppm).

1.16 Sulfonyl Azide (SZ17)

(SZ17) was prepared by two steps. First, the mixture of 5 (500 mg, 3.3mmol), 16 (5.20 mmol) and potassium carbonate (1.0 g, 7.2 mmol) inacetonitrile and water (9:1, mL), was stirred at room temperature for 12hours. After mixed with ethyl acetate (20 mL) and water (20 mL), thesystem was extracted by ethyl acetate (20 mL×3). The combined organicphase was dried by anhydrous sodium sulfate and concentrated.Intermediate 17 (330 mg, 37%) was obtained by flash chromatography(hexane:EtOAc=1:1-1:3; Rf=0.2 in hexane:EtOAc=1:1). ¹H-NMR (400 MHz,CDCl₃) δ: 7.37-7.16 (m, 6H), 6.91-6.89 (m, 2H), 6.81 (d, J=8.4 Hz, 1H),3.7-3.77 (m, 5H), 3.09 (t, J=6.4 Hz, 2H), 2.87 (t, J=6.0 Hz, 2H), 1.72(s, 1H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 159.5, 141.6, 135.6, 129.3,129.1, 128.6, 125.8, 120.1, 113.2, 112.2, 54.8, 53.1, 47.3, 33.9 ppm.

(SZ17) was prepared starting from 17 and known (SZ8) using the proceduredescribed for the preparation of (SZ9) in 54% yield. ¹H-NMR (400 MHz,CDCl₃) δ: 7.86 (d, J=8.0 Hz, 2H), 7.60 (d, J=8.0 Hz, 2H), 7.27-7.13 (m,6H), 6.99-6.95 (m, 2H), 6.81 (dd, J=8.0, 2.0 Hz, 1H), 3.81 (s, 3H), 3.69(s, 2H), 3.63 (s, 2H), 3.07 (t, J=7.2 Hz, 2H), 2.76 (t, J=7.2 Hz, 2H)ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 159.5, 147.6, 140.1, 136.6, 136.0,129.4, 129.2, 128.7, 128.5, 127.7, 127.2, 125.7, 120.8, 114.2, 112.4,58.2, 57.5, 54.9, 52.6, 31.1 ppm.

1.17 Thio Acid (TA2)

A mixture of known 11 (Kobayashi et al., Synthesis 1985, 6-7, 671-2)(6.0 g, 23 mmol) and 12 N HCl (40 mL) was kept at refluxing temperatureovernight. The reaction mixture was slowly cooled down and whitecrystals precipitated. The flask was then cooled to 0° C. for 10 minutesand the mixture was quickly filtrated. The crystals were washed withdichloromethane yielding the known product 12 (4.5 g, 73.2%). ¹H-NMR(250 MHz, CDCl₃) δ: 7.94 (d, J=6.2 Hz, 2H), 7.67 (bs, 2H), 3.43 (bs,4H), 1.69 (bs, 4H), 1.01 (s, 6H) ppm. HRMS (ESI⁺) for [M+H]⁺;calculated: 234.14866, found: 234.14797 (error m/z=−3.79 ppm).

Oxalyl chloride (740 mg, 0.5 mL, 5.83 mmol) was added dropwise into asolution of 12 (500 mg, 1.86 mmol) in dichloromethane at 0° C. Themixture was then stirred at room temperature for 8 hours. Then,dimethylthioformamide (0.6 mL, 6.7 mmol) was added to the abovesolution, and hydrogen sulfide was passed through the reaction mixturefor 10 minutes at a moderate rate. The course of the reaction wasmonitored by TLC and once compound 13 completely disappeared, theaddition of hydrogen sulfide was stopped. An excess of hexanes was addeduntil a yellow powder precipitated. The powder was collected byfiltration and product (TA2) (400 mg, 75% yield) was obtained by quickflash chromatography (DCM:MeOH=30:1). Rf=0.65 (DCM:MeOH=18:1) in theabsence of light. ¹H-NMR (400 MHz, CDCl₃) δ: 7.75 (d, J=8.4 Hz, 2H),6.80 (d, J=8.4 Hz, 2H), 4.32 (bs, 1H), 3.36-3.33 (m, 4H), 1.48-1.45 (m,4H), 0.98 (s, 6H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 187.7, 154.6, 130.1,125.2, 112.7, 43.8, 37.8, 28.6, 27.7 ppm. HRMS (ESI⁺) for [M+H]⁺;calculated: 250.12601, found: 250.12593 (error m/z=−0.33 ppm).

1.18 Thio Acid (TA3)

Compound 14 (300 mg, 1.26 mmol) was added slowly to a saturated solutionof sodium hydrogensulfide (211 mg, 3.76 mmol) in water at roomtemperature. The mixture was stirred at room temperature for 3 hours,ethyl acetate (50 mL) was then added followed by saturated aqueouspotassium carbonate solution (50 mL). After extraction by ethyl acetate(50 mL×3), the combined organic phases were dried over anhydrous sodiumsulfate and concentrated. The product (TA3) (200 mg, 67%) was obtainedby quick flash chromatography (hexanes:EtOAc=1:1). Rf=0.25 inhexane:EtOAc=1:1) in the absence of light. ¹H-NMR (400 MHz, CD₃OD) δ:6.36 (bs, 2H), 5.90 (bs, 3H), 1.76 (bs, 1H), 1.12 (s, 3H) ppm. ¹³C-NMR(100 MHz, CD₃OD) δ: 203.6, 168.8, 152.1, 142.4, 134.6, 131.5, 130.1,127.4, 17.6 ppm. HRMS (ESI⁺) for [M+H]⁺; calculated: 236.01983, found:236.01982 (error m/z=−0.04 ppm).

1.19 Thio Acid (TA4)

The synthesis of (TA4) was accomplished via the same procedure asdescribed for (TA3). ¹H-NMR (400 MHz, CDCl₃) δ: 7.35-7.15 (m, 5H), 6.66(s, 1H), 6.25 (s, 1H), 5.53 (s, 1H), 3.85 (s, 3H), 3.81 (bs, 2H), 3.65(s, 3H), 3.43-3.30 (m, 2H), 3.22-3.00 (m, 2H) ppm. ¹³C-NMR (100 MHz,CDCl₃) δ: 149.7, 148.8, 136.5, 130.6, 130.1, 129.5, 129.4, 123.6, 111.3,111.0, 66.3, 63.6, 56.2, 56.0, 45.4, 45.4 ppm. HRMS (ESI⁺) for [M]⁺;calculated: 344.13149, found: 344.13149 (error m/z=−0.02 ppm).

1.20 Thio Acid (TA5)

The synthesis of (TA5) was accomplished via the same procedure asdescribed for (TA2). ¹H-NMR (400 MHz, CDCl₃) δ: 7.08 (s, 1H), 7.07 (s,1H), 6.72 (s, 1H), 3.88 (s, 3H), 3.88 (s, 3H) ppm. ¹³C-NMR (100 MHz,CDCl₃) δ: 190.0, 160.7, 138.4, 106.1, 105.6, 55.5 ppm. HRMS (ESI⁺) for[M]⁺; calculated: 344.13149, found: 344.13149 (error m/z=−0.02 ppm).

1.21 Thio Acid (TA6)

To a solution of NaSH (90 mg, 1.6 mmol) in water (1 ml) was addeddropwise a solution of acid chloride in acetone (6 ml). The resultingmixture was stirred for 3 h. The solvent was removed under reducedpressure and resulting crude was basified using 10% NaOH solution(pH=12). The solution was slowly acidified using 2N HCl solution (pH=1).Corresponding thio acid (TA6) crashed out and was filtered, washed withdeionized water and dried under vacuum to obtain pale yellow crystals of(TA6). ¹H-NMR (400 MHz, CDCl₃) δ: 8.72-8.71 (m, 1H), 8.46-8.43 (m, 1H),8.2 (d, J=7.6 Hz, 1H), 7.68 (t, J=8 Hz, 1H) ppm. ¹³C-NMR (100 MHz,CDCl₃) δ: 188.2, 137.9, 133.4, 130.3, 128.3, 122.9 ppm.

1.22 Thio Acid (TA7)

The synthesis of (TA7) was accomplished via the same procedure asdescribed for (TA6). ¹H-NMR (400 MHz, CDCl₃) δ: 8.59-8.52 (m, 1H), 8.34(d, J=7.2 Hz, 1H), 8.08 (m, 1H), 7.88 (m, 1H), 7.57 (m, 3H) ppm. ¹³C-NMR(100 MHz, CDCl₃) δ: 188.1, 134.3, 133.4, 129.5, 129.0, 128.8, 128.65,127.3, 127.0, 125.4, 124.7 ppm.

Example 2 Reaction/Incubation Procedures and LC/MS Measurements

2.1 General Procedure for Incubations of Bcl-X_(L) with ReactiveFragments

In a 96-well plate, one thio acid building block (1 μL of a 2 mMsolution in methanol) and one sulfonyl azide building block (1 μL of a 2mM solution in methanol) were added to a solution of Bcl-X_(L) (98 μL ofa 2 μM Bcl-X_(L) solution in buffer (58 mM Na₂HPO₄, 17 mM NaH₂PO₄, 68 mMNaCl, 1 mM NaN₃, pH=7.40)). The 96-well plate was sealed and incubatedat 38.5° C. for six hours. The incubation samples were then subjected toliquid chromatography combined with mass spectrometry analysis in theselected ion mode (LC-MS-SIM, Zorbax SB-C18 preceded by a Phenomenex C18guard column, electrospray ionization and mass spectroscopic detectionin the positive selected ion mode, tuned to the expected molecular massof the product). The TGS hit compound was identified by the mass andretention time. As a control, identical building block combinations wereincubated in buffer without Bcl-X_(L) and subjected to LC/MS-SIManalysis. Comparison of the LC-MS-SIM chromatograms of these controlincubations with the chromatograms of the Bcl-X_(L) containingincubations allows to determine whether the protein is templating thecorresponding amidation reactions or not. For the Bcl-X_(L) containingincubation sample showing acylsulfonamide formation, a second controlhas been undertaken. Synthetically prepared acylsulfonamide wassubjected to LC/MS-SIM analysis and the retention time was compared withthe retention time identified in the Bcl-X_(L) containing incubation.

The gradient used for LC/MS-SIM is shown below:

Time B * Flowrate 0  10% 0.7 mL/min 4  20% 0.7 mL/min 12 100% 0.7 mL/min13 100% 0.7 mL/min 13.01 100% 1.5 mL/min 15.00 100% 1.5 mL/min 15.50 20% 1.5 mL/min 16.50  20% 1.0 mL/min 16.51  20%   0 mL/min * eluant A:H₂O (0.05% TFA); eluant B: CH₃CN 0.05% TFA)

2.2 Incubations at Various Bcl-X_(L) Concentrations

Different concentrations of Bcl-X_(L) were explored to determine theideal protein concentrations for the incubations with building blocks(SZ4) and (TA2). The minimal protein concentration for obtaining a goodratio between templated and non-templated reactions is 2 μM. Incubationsat higher concentrations give only slightly better ratios betweentemplated and non-templated reactions. Hence, we determined 2 μMBcl-X_(L) to be the most economical with regard to the proteinconsumption.

2.3 Comparison Between Incubations of SZ4 and TA2 Measured by theLC/MS-SIM and the LC/MS-Scan Mode

The sensitivity of the LC/MS can be significantly increased by utilizingthe MS instrument in the selected ion monitoring (LC/MS-SIM). Theadvantage of LC/MS-SIM over LC/MS-Scan for kinetic TGS has beenpreviously reported (Manetsch et al., J. Am. Chem. Soc. 2004; 126,12809-12818). See FIG. 4.

2.4 TGS Screening Criteria and Two Examples of TGS Incubation SamplesFailing at Templating the Formation of Acylsulfonamides

Examples of incubation samples failing at templating the formation ofacylsulfonamides are depicted in FIG. 5. To determine whether a buildingblock combination is a TGS hit or not, the ratio between the peak areasof the Bcl-X_(L)-templated reaction over the peak area of the incubationwithout Bcl-X_(L) is calculated. If this ratio is greater than 4, weconsider this particular combination to be a TGS hit combination.Further control incubation experiments (see FIG. 1 in the communication,FIG. 6, FIG. 8, FIG. 9, and FIG. 10) are performed to fully validatethis particular TGS hit.

2.5 Incubations of (SZ4) and (TA2) with Bovine Erythrocyte CarbonicAnhydrase II, Concanavalin A and Mouse Acetylcholinesterase.

The building blocks (SZ4) and (TA2) were incubated with proteins (20M)bovine erythrocyte carbonic anhydrase II (bCAII), concanavalin A (ConA)and mouse acetylcholinesterase (mAChE) respectively to test whetherthese proteins can also template the formation of acylsulfonamide(SZ4TA2). Incubations at 37° C. for 24 hours failed at yieldingpronounced amounts of (SZ4TA2).

2.6 Suppressing Bcl-X_(L)-Templated Incubations with Bak BH3 Peptide

Additional control experiments have been performed to test whether theBcl-X_(L)-templated reaction occurs at the BH3 binding site onBcl-X_(L). Reactive building blocks (SZ4) and (TA2) were incubated withBcl-X_(L) and pro-apoptotic Bak BH3 peptide. Bak is one of the naturalBcl-X_(L) ligands and theoretically competes with the reactive buildingblocks for binding on Bcl-X_(L) during the incubations. The influence ofBak BH3 peptide on the Bcl-X_(L)-templated reaction was studied atdifferent ratios of Bak BH3 peptide and Bcl-X_(L) (FIG. 10). At 20 μMBak BH3 peptide, the templated reaction is significantly suppressedcompared to the Bcl-X_(L) incubation without Bak BH3.

As an additional control experiment, the incubation of (SZ4) and (TA2)with Bak BH3 peptide was carried out for 24 hours (FIG. 7) demonstratingthat Bak BH3 can not template the formation of (SZ4TA2).

The sequence of the herein utilized Bak BH3 peptide is the following:

BakBH3 (wt): CMGQVGRQLAIIGDDINRRYDS

2.7 Suppressing Bcl-X_(L)-Templated Incubations with Bim, Mutant Bim andMutant Bak.

Additional control experiments have been performed with Bim, mutant Bim,and mutant Bak. Mutant Bim and mutant Bak are known to bind with loweraffinity towards Bcl-X_(L) compared to wildtype Bak BH3 and Bimpeptides. Therefore, the Bcl-X_(L) incubations containing mutant Bak BH3and mutant Bim display an increased amount of acylsulfonamide (SZ4TA2)compared to the Bcl-X_(L) incubations containing wild type Bak BH3 andBim peptides. The sequences of the various peptides are shown below:

BakBH3 (wt): CMGQVGRQLAIIGDDINRRYDS (see FIG. 7 and FIG. 10)BakBH3 (mt): CMGQVGRQAAIIGADINRRYDS BimBH3 (wt): CEIWIAQELRRIGDEFNAYYARBimBH3 (mt): CEIWIAQEARRIGAEFNAYYAR

2.8 Bcl-X_(L) Incubations Containing More than Two Reactive BuildingBlocks.

Initial experiments have been performed to investigate whether our TGSscreening via the amidation reaction can be performed with incubationscontaining more than two complimentary reactive building blocks.Previously, this has been shown to be applicable for standard in situligation chemistry approaches (Manetsch et al., supra; Krasinski et al.,J. Am. Chem. Soc. 2005, 127, 6686-6692). Our study revealed that theincubation sample containing 1 thioacid and 6 sulfonylazides (FIG. 11with (TA2), (SZ1)-(SZ6)) can give the same results as multipleincubations containing only two complimentary reacting building blocks.Attempts to run an incubation containing simultaneously 3 thioacids(TA1)-(TA3) and 6 azides (SZ1)-(SZ6) failed with 2 μM as well as 10 μMBcl-X_(L) concentrations.

Other positive incubations and their LC/MS-SIM measurements can be seenin the Figures. The concentration of active building blocks, proteinsand peptides are same as described above. The incubation reactiondetails are also as same as above. See FIGS. 12-19.

IC50 values for selected acylsulfonamides:

Example 3 Synthesis of a Cylsulfonamides

3.1 Acylsulfonamide (SZ7TA2)

Sodium boron hydride (60 mg, 1.5 mmol) was added slowly to the solutionof (SZ7) (450 mg, 1 mmol) in Methanol. The system was stirred for 30 minand removed all the solvent. Intermediate 24 was obtained by flashchromatography and used for next step directly. The solution of 24, 25(1 mmol), EDCI (2 mmol) and DMAP (0.2 mmol) in DCM was stirred for 12hours at room temperature, and the system was extracted by ethyl acetate(20 mL×3). The combined organic phase was dried by anhydrous sodiumsulfate and concentrated. And product (SZ7TA2) (102 mg, 16%) wasobtained by flash chromatography (hexane:EtOAc=1:1; Rf=0.2 inhexane:EtOAc=1:1). ¹H-NMR (400 MHz, CDCl₃) δ: 8.74 (s, 1H), 8.43 (s,1H), 8.24 (d, J=8.8 Hz, 1H), 7.95-7.89 (m, 3H), 7.67 (d, J=8.4 Hz, 2H),7.14 (d, J=6.4 Hz, 2H), 7.07 (d, J=6.8 Hz, 2H), 6.72 (d, J=8.8 Hz, 2H),5.46 (d, J=65.2 Hz, 1H), 3.26-3.25 (m, 4H), 3.13 (d, J=6.0 Hz, 2H), 2.96(d, J=6.4 Hz, 2H), 1.40-1.39 (m, 4H), 0.93 (s, 6H) ppm. ¹³C-NMR (100MHz, CDCl₃) δ: 164.1, 154.4, 138.4, 137.9, 136.5, 133.6, 131.2, 130.9,130.2, 130.0, 129.5, 129.4, 129.0, 126.9, 126.2, 125.4, 117.9, 113.0,43.7, 41.7, 37.8, 33.9, 28.6, 27.7 ppm. HRMS (ESI⁺) for [M+H]⁺;calculated: 638.18116, found: 638.18097 (error m/z=−0.29 ppm).

3.2 Acylsulfonamide (SZ9TA5)

The solution of (SZ10) (1 mmol), 26 (1 mmol), EDCI (2 mmol) and DMAP(0.2 mmol) in DCM was stirred for 12 hours at room temperature, and thesystem was extracted by ethyl acetate (20 mL×3). The combined organicphase was dried by anhydrous sodium sulfate and concentrated. Andproduct (SZ9TA5) (0.5 mmol, 50%) was obtained by flash chromatography(hexane:EtOAc=1:1; Rf=0.2 in hexane:EtOAc=1:1). ¹H-NMR (400 MHz, CDCl₃)δ: 7.97 (d, J=7.2 Hz, 2H), 7.78 (d, J=8.0 Hz, 2H), 7.46 (d, J=7.6 Hz,2H), 7.28-6.99 (m, 10H), 6.42 (s, 1H), 3.64-3.56 (m, 10H), 2.98-2.94 (m,2H), 2.63 (bs, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 160.7, 147.3, 137.2,136.0, 129.8, 129.2, 129.0, 127.7, 126.4, 106.6, 105.3, 58.1, 57.9,55.6, 52.9, 31.5 ppm. HRMS (ESI⁺) for [M+H]⁺; calculated: 682.14584,found: 682.14395 (error m/z=−2.77 ppm).

3.3 Acylsulfonamide (SZ10TA2) (and Acylsulfonamide (SZ9TA2))

(SZ9TA2) was prepared starting from (SZ10) and known 25 using theprocedure described for the preparation of (SZ9TA5) in 40% yield. ¹H-NMR(400 MHz, CDCl₃) δ: 8.09 (d, J=8.4 Hz, 2H), 7.85 (d, J=8.0 Hz, 2H), 7.63(d, J=8.6 Hz, 2H), 7.56 (d, J=8.4 Hz, 2H), 7.51 (d, J=8.0 Hz, 2H),7.24-7.16 (m, 7H), 6.86 (d, J=9.2 Hz, 2H), 3.77 (d, J=4.8 Hz, 4H), 3.32(t, J=4.8 Hz, 4H), 3.06 (t, J=7.2 Hz, 2H), 2.79 (t, J=7.2 Hz, 2H), 1.47(t, J=5.6 Hz, 4H), 0.98 (s, 6H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 154.1,147.0, 137.0, 135.7, 130.0, 129.5, 129.0, 128.9, 128.7, 128.3, 127.5,126.1, 113.1, 57.9, 57.7, 52.7, 43.9, 37.8, 31.3, 28.5, 27.7, 27.6 ppm.HRMS (ESI⁺) for [M+H]⁺; calculated: 733.22951, found: 733.22965 (errorm/z=0.19 ppm).

(SZ10TA2) was prepared starting from (SZ9TA2) using the proceduredescribed for the preparation of compounds 24 in 86% yield. ¹H-NMR (400MHz, CDCl₃) δ: 7.92 (d, J=7.2 Hz, 2H), 7.85 (d, J=7.2 Hz, 2H), 7.79 (d,J=7.2 Hz, 2H), 7.46 (d, J=7.6 Hz, 2H), 7.37 (d, J=7.6 Hz, 2H), 7.14-7.02(m, 5H), 6.81 (d, J=8.0 Hz, 2H), 3.57 (d, J=10.8 Hz, 4H), 3.34-3.30 (m,4H), 3.00 (t, J=7.2 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.44 (t, J=5.2 Hz,4H), 0.95 (s, 6H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 155.3, 145.6, 144.6,143.6, 137.5, 130.4, 130.0, 129.8, 129.7, 128.3, 127.2, 126.9, 114.8,62.9, 58.9, 54.0, 45.8, 39.2, 29.5, 28.3 ppm. HRMS (ESI⁺) for [M+H]⁺;calculated: 707.23901, found: 707.23934 (error m/z=0.46 ppm).

3.4 Acylsulfonamide (SZ15TA3)

Sodium boron hydride (60 mg, 1.5 mmol) was added slowly to the solutionof (SZ15) (1 mmol) in Methanol. The system was stirred for 30 min andremoved all the solvent. Intermediate 27 was obtained by flashchromatography and used for next step directly. The solution of 27, 28(1 mmol), EDCI (2 mmol) and DMAP (0.2 mmol) in DCM was stirred for 12hours at room temperature, and the system was extracted by ethyl acetate(20 mL×3). The combined organic phase was dried by anhydrous sodiumsulfate and concentrated. And product (SZ15TA3) (0.5 mmol, 50%) wasobtained by purification on preparative HPLC. ¹H-NMR (400 MHz, CDCl₃) δ:9.95 (s, 2H), 7.99 (d, J=7.5 Hz, 2H), 7.77 (d, J=7.5 Hz, 2H), 7.53 (d,J=7.5 Hz, 2H), 7.35-7.12 (m, 10H), 6.92 (t, J=7.5 Hz, 1H), 4.23 (bs,4H), 3.14-3.12 (m, 4H), 2.57 (s, 3H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ:169.8, 160.3 (d, ¹J_(CF)=150 Hz), 160.2, 139.9, 136.9 (d, ²J_(CF)=12.5Hz), 132.8, 132.5 (d, ³J_(CF)=10 Hz), 131.9, 131.7, 131.0, 130.8, 129.4,129.2, 129.2, 127.6, 127.0, 126.3, 122.0, 117.1, 116.8, 114.9 (d,²J_(CF)=21.5 Hz), 57.5, 52.7, 48.9, 28.8, 17.7 ppm. HRMS (ESI⁺) for[M+H]⁺; calculated: 666.11164, found: 666.10968 (error m/z=−2.94 ppm).

3.5 Acylsulfonamide (SZ15TA8)

The solution of 27, 29 (1 mmol), EDCI (2 mmol) and DMAP (0.2 mmol) inDCM was stirred for 12 hours at room temperature, and the system wasextracted by ethyl acetate (20 mL×3). The combined organic phase wasdried by anhydrous sodium sulfate and concentrated. And product(SZ15TA8) (0.82 mmol, 82%) was obtained by purification on preparativeHPLC. ¹H-NMR (400 MHz, CDCl₃) δ: 10.9 (bs, 1H), 8.04 (d, J=8.0 Hz, 2H),7.90 (d, J=8.4 Hz, 2H), 7.61 (d, J=8.0 Hz, 2H), 7.23-7.09 (m, 9H), 6.93(t, J=8.4 Hz, 1H), 4.27 (s, 2H), 4.25 (s, 2H), 3.23-3.16 (m, 4H) ppm.¹³C-NMR (100 MHz, CDCl₃) δ: 164.1, 162.6 (d, ¹J_(CF)=250 Hz), 161.2 (d,²J_(CF)=37 Hz), 152.7, 139.6, 137.6, 136.6, 133.0, 132.1 (d, ³J_(CF)=9.5Hz), 130.8, 130.3, 129.4, 129.2, 129.0, 127.3, 126.1, 121.4, 120.3,118.8, 117.5, 117.4, 114.6 (d, ²J_(CF)=22.6 Hz), 57.4, 52.7, 48.8, 28.7ppm. HRMS (ESI⁺) for [M+H]⁺; calculated: 653.09532, found: 653.09349(error m/z=−2.79 ppm).

3.6 Acylsulfonamide (SZ16TA6)

Compound 30 was prepared starting from (SZ16) using the proceduredescribed for the preparation of compounds 27, and (SZ16TA6) wasprepared starting from 30 and 31 using the procedure described for thepreparation of compounds (SZ15TA8) in 46% yield. ¹H-NMR (400 MHz, CD₃CN)δ: 8.58 (s, 1H), 8.36 (d, J=8.0 Hz, 1H), 8.15 (d, J=7.6 Hz, 1H), 8.10(d, J=6.8 Hz, 1H), 8.02 (d, J=7.6 Hz, 2H), 7.69-7.65 (m, 2H), 7.62 (d,J=8.0 Hz, 2H), 7.28 (t, J=10 Hz, 1H), 7.17-7.14 (m, 4H), 7.09 (dd,J=8.0, 4.0 Hz, 1H), 3.97 (s, 2H), 3.93 (s, 2H), 3.15 (t, J=7.2 Hz, 2H),2.86 (t, J=7.2 Hz, 2H) ppm. ¹³C-NMR (100 MHz, CD₃CN) δ: 163.8, 155.1 (d,¹J_(CF)=261 Hz), 148.4, 142.3, 138.9, 137.4 (d, ³J_(CF)=9.1 Hz), 135.1,134.5, 133.3 (d, ²J_(CF)=21.4 Hz), 130.4, 129.3, 129.2, 128.7, 127.8,127.4, 126.5, 123.4, 118.7 (d, ²J_(CF)=21.3 Hz), 117.6, 57.2, 56.5,52.2, 29.2 ppm.

3.7 Acylsulfonamide (SZ16TA8)

(SZ16TA8) was prepared starting from 30 and 29 using the proceduredescribed for the preparation of compounds (SZ15TA8) in 36% yield.¹H-NMR (400 MHz, CD₃CN) δ: 8.18 (dd, J=6.8, 1.6 Hz, 1H), 8.04 (d, J=8.0Hz, 2H), 7.90 (d, J=8.8 Hz, 2H), 7.75 (dd, J=4.4, 2.4 Hz, 1H), 7.64 (d,J=8.4 Hz, 2H), 7.37 (d, J=8.8 Hz, 2H), 7.34-7.31 (m, 1H), 7.20-7.15 (m,4H), 4.20 (s, 2H), 4.17 (s, 2H), 3.23-3.19 (m, 2H), 3.03-2.99 (m, 2H),2.62 (t, J=7.2 Hz, 2H), 1.44 (t, J=5.2 Hz, 4H), 0.95 (s, 6H) ppm.¹³C-NMR (100 MHz, CD₃CN) δ: 164.3, 155.6 (d, ¹J_(CF)=263 Hz), 152.6,139.9, 138.8, 138.3 (d, ³J_(CF)=10 Hz), 134.0, 131.2, 130.9, 130.5,130.0, 129.4, 128.9, 128.5, 127.1, 120.9, 119.1 (d, ²J_(CF)=21.4 Hz),57.0, 56.4, 51.7, 28.3 ppm.

3.8 Acylsulfonamide (SZ17TA7)

Compound 33 was prepared starting from (SZ17) using the proceduredescribed for the preparation of compounds 27, and (SZ17TA7) wasprepared starting from 33 and 32 using the procedure described for thepreparation of compounds (SZ15TA8) in 36% yield. HRMS (ESI⁺) for[M+H₂O]⁺; calculated: 614.19037, found: 614.18830 (error m/z=−3.36 ppm).

Example 4 Preparation of Other Compounds

4.1 Acylsulfonamide (SZ2TA1)

Ammonia gas was passed through a solution of compound 1 (1 g, 3.7 mmol)in DCM (100 mL) at 0° C. for 10 minutes. Brine (20 mL) was added. Theseparated organic phase was dried over anhydrous sodium sulfate andconcentrated. The product 17 (900 mg, 96.8%) was isolated by flashchromatography (hexanes:EtOAc=2:1). ¹H-NMR (250 MHz, Acetone-d6) δ: 7.91(d, J=10.0 Hz, 2H), 7.67 (d, J=10.0 Hz, 2H), 6.63 (bs, 2H), 4.74 (s, 2H)ppm.

A mixture of 17 (100 mg, 0.40 mmol), 4 (54.3 mg, 0.40 mmol) andpotassium carbonate (100 mg, 0.72 mmol) in acetonitrile and water (9:1)was stirred at room temperature for 12 hours. To this mixture, ethylacetate (20 mL) and water (20 mL) were added, and the resulting biphasicmixture was extracted by ethyl acetate (20 mL×3). The combined organicphases were dried over anhydrous sodium sulfate and concentrated.Product 18 (108 mg, 88.5%) was obtained by flash chromatography(hexane:EtOAc=1:1; Rf=0.2 in hexane:EtOAc=1:1). ¹H-NMR (250 MHz, CDCl₃)δ: 7.73 (d, J=10.0 Hz, 2H), 7.30 (d, J=10.0 Hz, 2H), 7.22-7.07 (m, 5H),5.18 (bs, 2H), 3.51 (s, 2H), 2.76-2.70 (m, 2H), 2.60-2.54 (m, 2H), 2.19(s, 3H) ppm. ¹³C-NMR (62.5 MHz, CDCl₃) δ: 144.6, 140.6, 140.2, 129.4,128.7, 128.4, 126.4, 126.1, 61.6, 59.1, 42.2, 33.8 ppm. HRMS (ESI⁺) for[M+H]⁺; calculated: 305.1324, found: 305.1325. (error m/z=0.3 ppm).

A mixture of 34 (31 mg, 0.25 mmol), 18 (76 mg, 0.25 mmol), EDCI (60 mg,0.314 mmol) and DMAP (8 mg, 0.065 mmol) in dichloromethane (10 mL) wasstirred for 19 h. Product (SZ2TA1) (87 mg, 85%) was isolated after flashchromatography (DCM: MeOH=18:1). Rf=0.68 (DCM: MeOH=4:1). ¹H-NMR (400MHz, DMSO-d6) δ: 7.86 (d, J=7.6 Hz, 4H), 7.48 (d, J=8 Hz, 2H), 7.37-7.19(m, 8H), 4.16 (bs, 2H), 3.09-3.07 (m, 2H), 2.94-2.90 (m, 2H), 2.58 (s,3H) ppm. ¹³C-NMR (100 MHz, DMSO-d6) δ: 169.7, 146.2, 138.4, 131.2,130.6, 129.9, 129.4, 129.2, 129.0, 128.3, 128.0, 127.3, 59.5, 57.4, 31.1ppm. HRMS (ESI⁺) for [M+H]⁺; calculated: 409.1586, found: 409.1582(error m/z=−0.9 ppm).

4.2 Acylsulfonamide (SZ2TA2)

A solution of 15 (58 mg, 0.25 mmol), 18 (76 mg, 0.25 mmol), EDCI (60 mg,0.314 mmol) and DMAP (8 mg, 0.065 mmol) in dichloromethane (10 mL) wasstirred for 19 hours. Product (SZ2TA2) (42 mg, 32.5%) was isolated afterflash chromatography (DCM: MeOH=24:1). Rf=0.6 (DCM: MeOH=6:1). ¹H-NMR(400 MHz, DMSO-d6) δ: 7.85 (d, J=8 Hz, 2H), 7.69 (d, J=8.8 Hz, 2H), 7.44(d, J=8 Hz, 2H), 7.24-7.16 (m, 6H), 6.84 (d, J=8.8 Hz, 2H), 3.80 (bs,2H), 3.27-3.24 (m, 4H), 3.14 (s, 2H), 2.79 (bs, 4H), 2.33 (s, 3H),1.35-1.32 (m, 2H), 0.91 (s, 6H) ppm. ¹³C-NMR (100 MHz, DMSO-d6) δ:166.9, 153.9, 139.9, 130.7, 129.9, 129.3, 129.0, 128.1, 126.8, 126.3,113.5, 60.5, 58.5, 44.5, 41.7, 38.1, 32.6, 29.1, 28.3, 28.2 ppm. HRMS(ESI⁺) for [M+H]⁺; calculated: 520.2634, found: 520.2627 (error m/z=1.3ppm).

4.3 Acylsulfonamide (SZ2TA3)

Compound 20 (500 mg, 2.1 mmol) was added into 1M NaOH and then stirredovernight. The resulting mixture was treated with 2N HCl. Product 21(310 mg, 67%) can be filtered out and dried. The crude product was usedfor next step directly.

A solution of 21 (45 mg, 0.205 mmol), 18 (61 mg, 0.205 mmol), EDCI (60mg, 0.314 mmol) and DMAP (8 mg, 0.065 mmol) in dichloromethane (10 mL)was stirred for 24 h. Product (SZ2TA3) (58 mg, 55%) was isolated bypreparative HPLC. (DCM: MeOH=36:1). Rf=0.55 (DCM: MeOH=8:1). ¹H-NMR (250MHz, Acetone-d6) δ: 8.03 (d, J=8 Hz, 2H), 7.85-7.78 (m, 4H), 7.39-7.36(m, 3H), 7.14-7.11 (m, 5H), 4.52 (bs, 2H), 3.36-3.32 (m, 2H), 3.12-3.09(m, 2H), 2.84 (s, 3H), 2.48 (s, 3H) pm. ¹³C-NMR (100 MHz, CDCl₃) δ:169.7, 161.2, 159.8, 140.9, 135.1, 134.4, 131.8, 131.6, 131.5, 129.2,129.1, 129.0, 128.6, 127.5, 126.9, 122.4, 59.3, 57.5, 39.6, 30.5, 17.5ppm. HRMS (ESI⁺) for [M+H]⁺; calculated: 506.1572, found: 506.1565(error m/z=−1.4 ppm).

4.4 Acylsulfonamide (SZ4TA1)

A solution of (TA1) (50 mg, 0.36 mmol), (SZ4) (136 mg, 0.36 mmol) and2,6-lutidine (40 mg, 0.37 mmol) in chloroform (10 mL) was stirred at 70°C. for 16 h. Chloroform (30 mL) was added to the reaction and theresulting mixture was washed sequentially with saturated copper sulfateaqueous solution (50 mL) and brine (20 mL). The organic phase was thendried over anhydrous sodium sulfate and concentrated. Product (SZ4TA1)(89 mg, 54%) was isolated after flash chromatography (DCM: MeOH=26:1).Rf=0.58 (DCM: MeOH=8:1). ¹H-NMR (400 MHz, CDCl₃) δ: 8.93 (s, 1H), 8.87(d, J=1.2 Hz, 1H), 8.68 (s, 1H), 8.13 (d, J=8.4 Hz, 1H), 7.76 (d, J=7.6Hz, 2H), 7.56 (t, J=7.2 Hz, 1H), 7.45-7.37 (m, 4H), 7.29-7.22 (m, 4H),6.81 (d, J=8.8 Hz, 1H), 3.58-3.54 (m, 2H), 3.20-3.17 (m, 2H) ppm.¹³C-NMR (100 MHz, CDCl₃) δ: 164.3, 147.6, 135.5, 133.7, 131.2, 130.9,129.3, 129.0, 127.7, 127.4, 124.5, 113.7, 42.1, 33.2 ppm. HRMS (ESI⁺)for [M+H]⁺; calculated: 458.08389, found: 458.08350 (error m/z=−0.84ppm).

4.5 Acylsulfonamide (SZ5TA1)

A solution of (TA1) (50 mg, 0.36 mmol), (SZ5) (71 mg, 0.36 mmol) and2,6-lutidine (40 mg, 0.37 mmol) in chloroform (10 mL) was stirred at 70°C. for 16 h. Chloroform (30 mL) was added to the reaction and theresulting mixture was washed sequentially with saturated copper sulfateaqueous solution (50 mL) and brine (20 mL). The organic phase was thendried over anhydrous sodium sulfate and concentrated. Product (SZ5TA1)(67 mg, 68%) was isolated after flash chromatography (DCM: MeOH=38:1).Rf=0.7 (DCM: MeOH=10:1). ¹H-NMR (250 MHz, CDCl₃) δ: 9.62 (s, 1H), 7.95(d, J=8 Hz, 2H), 7.75 (d, J=8.0 Hz, 2H), 7.43 (t, J=7.2 Hz, 1H),7.32-7.22 (m, 4H), 2.32 (s, 3H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 164.5,145.1, 135.4, 133.4, 129.5, 128.7, 128.5, 127.9, 127.8, 21.6 ppm. HRMS(ESI) for [M+H]⁺; calculated: 276.0694, found: 276.0696 (error m/z=−0.7ppm).

4.6 Acylsulfonamide (SZ5TA2)

A solution of 15 (58 mg, 0.25 mmol), 22 (43 mg, 0.25 mmol), EDCI (60 mg,0.314 mmol) and DMAP (8 mg, 0.065 mmol) in dichloromethane (10 mL) wasstirred for 30 h. Product (SZ5TA2) (68 mg, 70%) was isolated after flashchromatography (DCM: MeOH=16:1). Rf=0.6 (DCM: MeOH=8:1). ¹H-NMR (400MHz, CDCl₃) δ: 8.70 (bs, 1H), 8.00 (d, J=8 Hz, 2H), 7.63 (d, J=8.4 Hz,2H), 7.30 (d, J=8 Hz, 2H), 6.82 (d, J=7.6 Hz, 2H), 3.30 (t, J=6 Hz, 4H),2.40 (s, 3H), 1.46 (bs, 4H), 0.97 (s, 6H) ppm. ¹³C-NMR (100 MHz,DMSO-d6) δ: 165.1, 154.4, 144.5, 137.9, 130.9, 130.0, 128.3, 119.2,113.4, 43.9, 38.0, 29.2, 28.3, 21.9 ppm. HRMS (ESI⁺) for [M+H]⁺;calculated: 387.1742, found: 387.1742 (error m/z=−1.8 ppm).

4.7 Acylsulfonamide (SZ9TA1)

(SZ9TA1) was prepared starting from 30 and 34 using the proceduredescribed for the preparation of compounds (SZ15TA8) in 37% yield.¹H-NMR (400 MHz, CDCl₃) δ: 8.03 (d, J=8.4 Hz, 2H), 7.78 (d, J=8.4 Hz,2H), 7.71 (d, J=7.6 Hz, 2H), 7.51-7.46 (m, 4H), 7.41-7.33 (m, 3H),7.19-7.04 (m, 5H), 3.61 (s, 4H), 2.99 (t, J=7.2 Hz, 2H), 2.68 (t, J=7.2Hz, 2H) ppm. ¹³C-NMR (100 MHz, CD₃CN) δ: 170.0, 164.3, 146.9, 145.6,137.4, 137.2, 135.7, 133.5, 131.2, 130.1, 129.5, 129.1, 128.9, 128.8,128.4, 127.7, 127.6, 126.3, 58.0, 57.9, 52.8, 31.5 ppm. HRMS (ESI⁺) for[M+H]⁺; calculated: 622.12471, found: 622.12402 (error m/z=−1.10 ppm).

4.8 Acylsulfonamide (SZ10TA1)

Sodium boron hydride (60 mg, 1.5 mmol) was added slowly to the solutionof (SZ9TA1) (1 mmol) in Methanol. The system was stirred for 30 min andremoved all the solvent. (SZ10TA1) was obtained by flash chromatography(hexane:EtOAc=1:1; Rf=0.15 in hexane:EtOAc=1:1). ¹H-NMR (400 MHz, CD₃OD)δ: 8.04 (d, J=8.0 Hz, 2H), 7.92 (d, J=7.6 Hz, 2H), 7.79 (d, J=7.6 Hz,2H), 7.44-7.27 (m, 7H), 7.12-6.97 (m, 5H), 3.55 (s, 2H), 3.49 (s, 2H),2.96 (t, J=7.2 Hz, 2H), 2.57 (t, J=7.2 Hz, 2H) ppm. ¹³C-NMR (100 MHz,CD₃OD) δ: 173.4, 144.3, 143.6, 142.4, 137.0, 136.3, 131.5, 129.2, 129.0,128.8, 128.7, 128.6, 127.7, 126.5, 126.0, 125.7, 57.7, 52.7, 30.7 ppm.HRMS (ESI) for [M+H]⁺; calculated: 596.13421, found: 596.13388 (errorm/z=−0.55 ppm).

4.9 Acylsulfonamide (SZ10TA5)

(SZ10TA5) was prepared starting from (SZ9TA5) using the proceduredescribed for the preparation of compounds (SZ10TA1) in 91% yield.¹H-NMR (400 MHz, CDCl₃) δ: 7.96 (d, J=8.0 Hz, 2H), 7.80 (d, J=8.04 Hz,2H), 7.50-7.44 (m, 4H), 7.15-7.01 (m, 7H), 6.57 (d, J=1.6 Hz, 1H), 3.71(s, 6H), 3.60 (s, 2H), 3.58 (s, 2H), 3.31-3.30 (m, 2H), 2.64-2.60 (m,2H) ppm. ¹³C-NMR (100 MHz, CD₃CN) δ: 171.0, 162.0, 145.7, 145.2, 143.5,141.6, 138.0, 137.4, 130.6, 130.4, 129.9, 129.8, 128.7, 127.1, 126.9,107.3, 105.6, 58.8, 56.1, 55.9, 53.9, 31.8 ppm. HRMS (ESI⁺) for [M+H]⁺;calculated: 656.15534, found: 656.15466 (error m/z=−1.03 ppm).

4.10 Acylsulfonamide (SZ15TA1)

(SZ15TA1) was prepared starting from 27 and 34 using the proceduredescribed for the preparation of (SZ15TA3) in 61% yield. ¹H-NMR (400MHz, CD₃CN) δ: 8.07 (d, J=8.4 Hz, 2H), 7.82 (d, J=7.6 Hz, 2H), 7.21 (d,J=8.0 Hz, 2H), 7.61 (t, J=7.2 Hz, 1H), 7.47 (t, J=7.6 Hz, 2H), 7.38-7.34(m, 1H), 7.26-7.12 (m, 6H), 7.07 (t, J=8.8 Hz, 1H), 4.40 (s, 2H), 4.32(s, 2H), 3.35-3.31 (m, 2H), 3.25-3.21 (m, 2H) ppm. ¹³C-NMR (100 MHz,CD₃CN) δ: 165.9, 162.7 (d, ¹J_(CF)=249 Hz), 160.5, 160.1, 140.9, 137.7,136.9, 134.1, 133.7, 133.4, 133.3 (d, ³J_(CF)=10 Hz), 132.0, 131.9,130.6, 129.9, 129.3, 128.9, 127.8, 126.7, 115.4 (d, ²J_(CF)=22.5 Hz),57.8, 53.4, 49.4, 28.3 ppm.

4.11 Acylsulfonamide (SZ15TA2)

(SZ15TA2) was prepared starting from 27 and 25 using the proceduredescribed for the preparation of (SZ15TA3) in 48.4% yield. ¹H-NMR (400MHz, CDCl₃) δ: 11.13 (bs, 1H), 7.96 (d, J=7.6 Hz, 2H), 7.79 (d, J=8.0Hz, 2H), 7.56 (d, J=8.0 Hz, 2H), 7.27-7.12 (m, 10H), 6.93 (t, J=8.8 Hz,1H), 4.27 (s, 2H), 4.24 (s, 2H), 3.35 (bs, 4H), 3.19 (bs, 2H), 3.15 (bs,2H), 1.61 (bs, 4H), 0.99 (s, 6H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 164.2,162.1 (d, ¹J_(CF)=241 Hz), 161.3, 160.8, 149.9, 140.0, 137.0, 136.7,132.9, 132.3 (d, ³J_(CF)=9.5 Hz), 130.8, 130.5, 130.3, 129.2, 129.0,127.4, 126.1, 125.8, 117.4, 114.7 (d, ²J_(CF)=22.6 Hz), 57.4, 52.8,48.8, 48.5, 36.6, 28.6, 27.8, 27.4 ppm.

4.12 Acylsulfonamide (SZ15TA4)

(SZ15TA4) was prepared starting from 27 and 35 using the proceduredescribed for the preparation of (SZ15TA3) in 48.4% yield. ¹H-NMR (400MHz, CDCl₃) δ: 8.56 (bs, 1H), 7.94 (d, J=8.0 Hz, 2H), 7.59 (d, J=8.0 Hz,2H), 7.35-7.30 (m, 4H), 7.24-7.18 (m, 10H), 6.98 (t, J=8.4 Hz, 1H), 6.65(s, 1H), 6.18 (s, 1H), 5.70 (s, 1H), 4.28 (bs, 4H), 4.09 (d, J=16.4 Hz,1H), 3.89 (d, J=16.8 Hz, 1H), 3.83 (s, 3H), 3.57 (s, 3H), 3.45 (bs, 2H),3.19-3.02 (m, 6H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 165.0, 162.4 (d,¹J_(CF)=251 Hz), 161.5, 149.8, 148.9, 139.8, 137.4, 137.0, 134.6, 133.2,132.5 (d, ³J_(CF)=9.5 Hz), 131.3, 130.9, 130.7, 130.6, 129.5, 129.0,127.7, 126.4, 122.9, 120.8, 117.5, 117.3, 115.0 (d, ²J_(CF)=22.6 Hz),111.0 (d, ²J_(CF)=14.9 Hz), 66.4, 57.8, 56.1, 56.0, 54.0, 52.9, 49.1,46.0, 29.0, 23.7 ppm. HRMS (ESI⁺) for [M+H]⁺; calculated: 774.22330,found: 774.22223 (error m/z=−1.37 ppm).

4.13 Acylsulfonamide (SZ15TA5)

(SZ15TA5) was prepared starting from 27 and 26 using the proceduredescribed for the preparation of (SZ15TA3) in 44.2% yield. ¹H-NMR (400MHz, CDCl₃) δ: 8.84 (bs, 1H), 8.04 (d, J=7.2 Hz, 2H), 7.60 (d, J=7.6 Hz,2H), 7.28 (dd, J=14.0, 7.2 Hz, 1H), 7.16 (bs, 6H), 6.97 (t, J=8.4 Hz,1H), 6.91 (s, 2H), 6.55 (s, 1H), 4.36 (s, 1H), 4.33 (s, 1H), 3.70 (s,6H), 3.22 (bs, 4H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 165.1, 162.5 (d,¹J_(CF)=226 Hz), 161.1, 140.3, 136.9, 136.2, 133.1, 133.0, 132.7, 131.3,131.1, 129.6, 127.9, 126.5, 116.4 (d, ²J_(CF)=16.8 Hz), 115.1 (d,²J_(CF)=22.5 Hz), 106.3, 106.0, 57.7, 55.8, 52.9, 49.1, 28.7 ppm. HRMS(ESI⁺) for [M+H]⁺; calculated: 629.13415, found: 629.13427 (errorm/z=0.20 ppm).

4.14 Acylsulfonamide (SZ15TA6)

(SZ15 TA6) was prepared starting from 27 and 31 using the proceduredescribed for the preparation of (SZ15TA3) in 60.7% yield ¹H-NMR (400MHz, CDCl₃) δ: 8.63 (s, 1H), 8.24 (d, J=8 Hz, 2H), 8.17 (d, J=7.6 Hz,2H), 8.05 (d, J=7 Hz, 3H), 7.64 (d, J=7 Hz, 2H), 7.54 (t, J=7 Hz, 1H),7.28-7.23 (m, 2H), 7.14 (s, 6H), 7.02-6.93 (m, 2H), 4.36 (s, 2H), 4.32(s, 2H), 3.24-3.21 (m, 4H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 163.5, 162.3(d, ¹J_(CF)=208 Hz), 160.8, 148.0, 139.8, 136.6, 134.1, 133.0, 132.6,131.0, 130.5, 129.9, 129.3, 127.5, 126.2, 123.4, 116.6 (d, ²J_(CF)=18Hz), 114.8 (d, ²J_(CF)=22 Hz), 57.5, 52.6, 48.8, 28.5 ppm. HRMS (ESI⁺)for [M+Na]⁺; calculated: 636.0800, found: 636.0804 (error m/z=0.53 ppm).

4.15 Acylsulfonamide (SZ15TA7)

(SZ15TA7) was prepared starting from 27 and 32 using the proceduredescribed for the preparation of (SZ15TA3) in 48.4% yield. ¹H-NMR (400MHz, CD₃OD) δ: 8.11 (d, J=8.4 Hz, 2H), 7.98 (d, J=8.4 Hz, 1H), 7.95 (d,J=8.8 Hz, 1H), 7.85 (d, J=8.4 Hz, 1H), 7.68-7.64 (m, 3H), 7.47 (d, J=8.0Hz, 1H), 7.43 (d, J=9.2 Hz, 1H), 7.39-7.28 (m, 2H), 7.23-7.10 (m, 6H),7.02 (t, J=8.8 Hz, 1H), 4.28 (s, 2H), 4.21 (s, 2H), 3.24-3.21 (m, 2H),3.12-3.09 (m, 2H) ppm. ¹³C-NMR (100 MHz, CD₃OD) δ: 168.1, 162.3 (d,¹J_(CF)=249.6 Hz), 140.2, 136.5, 133.9, 132.3, 132.0, 130.9, 130.6,130.5, 130.0, 129.2, 128.7, 127.3, 127.1, 126.9, 126.6, 126.0, 124.4 (d,³J_(CF)=10.7 Hz), 114.5 (d, ²J_(CF)=22.9 Hz), 57.4, 53.3, 48.5, 28.7ppm.

4.16 Acylsulfonamide (SZ15TA9)

(SZ15TA9) was prepared starting from 27 and 36 using the proceduredescribed for the preparation of (SZ15TA9) in 91.6% yield. ¹H-NMR (400MHz, CDCl₃) δ: 10.32 (s, 1H), 8.03 (d, J=8 Hz, 2H), 7.64 (d, J=8 Hz,2H), 7.28-7.22 (m, 3H), 7.14-7.12 (m, 7H), 6.94 (t, J=9 Hz, 1H), 4.45(s, 2H), 4.37 (s, 2H), 3.27 (s, 4H) ppm. ¹³C-NMR (100 MHz, CDCl₃) δ:162.0 (d, ¹J_(CF)=251 Hz), 160.9, 160.5, 155.1, 146.6, 144.7, 140.2,136.5, 135.3, 132.9 (d, ³J_(CF)=9.6 Hz), 132.0, 131.3, 130.7, 129.2,129.1, 127.7, 126.2, 118.5, 117.1, 115.5 (d, ²J_(CF)=17 Hz), 114.8 (d,²J_(CF)=22.6 Hz), 114.3, 112.8, 57.4, 52.6, 48.8, 28.1 ppm. HRMS (ESI⁺)for [M+H]⁺; calculated: 559.0923, found: 559.0915 (error m/z=−1.31 ppm).

4.17 Acylsulfonamide (SZ15TA10)

(SZ15TA10) was prepared starting from 27 and 37 using the proceduredescribed for the preparation of (SZ15TA9) in 91.6% yield. ¹H-NMR (400MHz, CDCl₃) δ: 8.30 (s, 1H), 7.98 (d, J=8 Hz, 2H), 7.83 (bs, 1H), 7.54(d, J=8 Hz, 2H), 7.18-7.10 (m, 7H), 6.99 (s, 1H), 6.91 (t, J=8.4 Hz,1H), 4.06 (s, 2H), 4.02 (s, 2H), 3.13-3.11 (m, 2H), 2.97-2.95 (m, 2H)ppm. ¹³C-NMR (100 MHz, CDCl₃) δ: 162.2 (d, ¹J_(CF)=250 Hz), 160.3,153.4, 151.2, 141.9, 137.9, 136.4, 134.2, 130.9 (d, ³J_(CF)=9.6 Hz),129.9, 129.7, 129.0, 128.7, 126.6, 125.8, 120.3 (d, ²J_(CF)=17.6 Hz),114.3 (d, ²J_(CF)=23 Hz), 108.7, 57.5, 52.9, 49.0, 29.6 ppm. HRMS (ESI⁺)for [M+H]⁺; calculated: 560.0875, found: 560.0873 (error m/z=−0.34 ppm).

4.18 Acylsulfonamide (SZ17TA3)

(SZ17TA3) was prepared starting from 33 and 28 using the proceduredescribed for the preparation of (SZ15TA9) in 79.5% yield. ¹H-NMR (400MHz, CDCl₃) δ: 9.81 (s, 1H), 8.05 (d, J=8 Hz, 2H), 7.79 (d, J=7.2 Hz,2H), 7.53 (d, J=7.6 Hz, 2H), 7.42-7.33 (m, 3H), 7.21-15 (m, 6H), 6.95(s, 1H), 6.87 (d, J=8 Hz, 1H), 6.81 (d, J=7.2 Hz, 1H), 4.29 (s, 2H),4.19 (s, 2H), 3.7 (s, 3H), 3.11-3.10 (m, 4H), 2.60 (s, 3H) ppm.

4.19 Acylsulfonamide (SZ3TA6)

(SZ3TA6) was prepared starting from 38 and 31 using the proceduredescribed for the preparation of (SZ15TA9) in 45.5% yield. ¹H-NMR (250MHz, acetone-d₆) δ: 8.61 (s, 1H), 8.31 (d, J=7.5 Hz, 1H), 8.22 (d, J=10Hz, 1H), 7.9 (d, J=7.5 Hz, 2H), 7.75-7.64 (m, 3H), 1.99 (s, 3H) ppm.¹³C-NMR (60 MHz, acetone-d₆) δ: 169.7, 149.2, 145.1, 135.2, 134.1,131.1, 130.5, 128.0, 123.9, 119.1, 24.3 ppm.

4.20 Acylsulfonamide (SZ3TA9)

(SZ3TA9) was prepared starting from 38 and 36 using the proceduredescribed for the preparation of (SZ15TA9) in 35.5% yield. ¹H-NMR (400MHz, CD₃OD) δ: 7.97 (d, J=8.8 Hz, 2H), 7.75 (d, J=8.8 Hz, 2H), 7.70 (d,J=1.2 Hz, 1H), 7.25 (d, J=1.2 Hz, 1H), 6.58 (dd, J=3.2, 1.6 Hz, 1H),2.13 (s, 3H) ppm. ¹³C-NMR (100 MHz, CD₃OD) δ: 170.9, 156.4, 147.1,145.6, 143.9, 133.8, 129.4, 118.9, 117.8, 112.4, 22.9 ppm. HRMS (ESI⁺)for [M+H]⁺; calculated: 309.05397, found: 309.05467 (error m/z=2.28ppm).

4.21 Acylsulfonamide (SZ9TA7)

(SZ9TA7) was prepared starting from (SZ10) and 32 using the proceduredescribed for the preparation of (SZ15TA9) in 69.3% yield. ¹H-NMR (400MHz, CDCl₃) δ: 8.44 (s, 1H), 7.96 (d, J=8.0 Hz, 2H), 7.73-7.71 (m, 3H),7.57-7.55 (m, 3H), 7.35 (d, J=8.0 Hz, 2H), 7.15-6.95 (m, 10H), 3.37 (s,2H), 3.31 (s, 2H), 2.86 (t, J=6.4 Hz, 2H), 2.50 (bs, 2H) ppm. ¹³C-NMR(100 MHz, CDCl₃) δ: 147.0, 143.5, 140.0, 136.8, 135.8, 133.4, 131.4,130.4, 129.5, 128.9, 128.8, 128.7, 128.0, 127.7, 127.4, 127.1, 126.9,126.1, 125.7, 124.4, 57.6, 57.4, 52.5, 31.2 ppm. HRMS (ESI⁺) for [M+H]⁺;calculated: 309.05397, found: 309.05467 (error m/z=2.28 ppm).

1. A process for the preparation of an acylsulfonamide (3), the processcomprising reacting a thioacid (1) with a sulfonyl azide (2) in thepresence of a protein of the Bcl-2 family, wherein the thioacid (1), thesulfonyl azide (2), and the acylsulfonamide (3) correspond to Formulae(1), (2), and (3):

Z₁ is hydrocarbyl, substituted hydrocarbyl, heteroaryl, or heterocyclo;and Z₂ is hydrocarbyl, substituted hydrocarbyl, heteroaryl, orheterocyclo.
 2. The process of claim 1 wherein Z₁ is aryl, substitutedaryl, or heteroaryl.
 3. The process of claim 1 wherein Z₁ has theformula:

wherein Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄ are independently hydrogen,hydroxyl, protected hydroxyl, halo, hydrocarbyl, substitutedhydrocarbyl, heterocyclo, heteroaryl, alkoxy, alkenoxy, alkynoxy,aryloxy, arylalkoxy (heterocyclo)alkoxy, trihaloalkoxy, amino, amido, orcyano, or two of Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄, together with the carbonatoms to which they are attached, form a fused carbocyclic (e.g.,napthyl) or heterocyclic ring.
 4. The process of claim 3 wherein Z₁₀,Z₁₁, Z₁₂, Z₁₃, and Z₁₄ are independently hydrogen, amino, alkoxy, nitro,or trihalomethoxy.
 5. The process of claim 1 wherein Z₁ has the formula:

wherein A is phenyl or a five- or six-membered aromatic carbocyclic orheterocyclic ring wherein from one to three carbon atoms may be replacedby a heteroatom selected from N, O, or S, and wherein A is substitutedwith Z₁₀₀ and Z₁₀₁ through ring carbon atoms or ring heteroatoms, andZ₁₀₀ and Z₁₀₁ are independently hydrogen, hydrocarbyl, substitutedhydrocarbyl, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy,heterocyclo(alkoxy), or halo.
 6. The process of claim 1 wherein Z₁ issubstituted or unsubstituted furyl, thienyl, pyridyl, oxazolyl,isoxazolyl, imidazolyl, pyridyl, pyrimidyl, purinyl, triazolyl, orthiazolyl.
 7. The process of claim 1 wherein Z₁ is substituted orunsubstituted morpholino, pyran, tetrahydropyran, piperazinyl,piperidinyl, tetrahydropyridinyl, pyrrolidinyl, pyrrolinyl,1,4-diazepanyl, or azepinyl.
 8. The process of claim 1 wherein Z₁ is—(CH₂)_(x)—Z₁₀₂ wherein Z₁₀₂ is hydrogen, hydroxyl, protected hydroxyl,heterocyclo, amino, amido, alkoxy, aryloxy, cyano, nitro, thiol, or anacetal, ketal, ester, ether, or thioether, and x is 1, 2, or
 3. 9. Theprocess of claim 1 wherein Z₁ is heteroaryl, heterocyclo, or has theformula:

wherein Z₁₀, Z₁₁, Z₁₂, Z₁₃, and Z₁₄ are independently hydrogen, amino,alkoxy, nitro, or trihalomethoxy (e.g., trifluoromethoxy); or Z₁ is—(CH₂)_(x)—Z₁₀₂ wherein Z₁₀₂ is hydrogen, hydroxyl, protected hydroxyl,heterocyclo, amino, amido, alkoxy, aryloxy, cyano, nitro, thiol, or anacetal, ketal, ester, ether, or thioether, and x is 1, 2, or
 3. 10. Theprocess of any one of claims 1-9 wherein Z₂ is substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, alkaryl, or aralkyl. 11.The process of any one of claims 1-9 wherein Z₂ has the formula:

wherein Z₂₀, Z₂₁, Z₂₂, Z₂₃, and Z₂₄ are independently hydrogen, halo,hydrocarbyl, substituted hydrocarbyl, alkoxy, alkenoxy, alkynoxy,aryloxy, nitro, cyano, amino, or amido, or two of Z₂₀, Z₂₁, Z₂₂, Z₂₃,and Z₂₄, together with the carbon atoms to which they are attached, forma fused carbocyclic or heterocyclic ring.
 12. The process of claim 11wherein Z₂₀, Z₂₁, Z₂₂, Z₂₃, and Z₂₄ are independently alkyl, substitutedalkyl, amino, alkoxy, alkenoxy, alkynoxy, or aryloxy.
 13. The process ofany one of claims 1-10 wherein Z₂ is phenyl, substituted phenyl,napthyl, or substituted napthyl.
 14. The process of any one of claims1-10 wherein Z₂ may be —(CH₂)_(x)—Z₂₀₀ wherein Z₂₀₀ is hydrogen,hydroxyl, protected hydroxyl, heterocyclo, amino, amido, alkoxy,aryloxy, cyano, nitro, thiol, or an acetal, ketal, ester, ether, orthioether, and x is 1, 2, or
 3. 15. The process of any one of claims 1-9wherein Z₂ is substituted or unsubstituted furyl, thienyl, pyrrolyl,oxazolyl, imidazolyl, pyridyl, pyrimidyl, purinyl, triazolyl, orthiazolyl.
 16. The process of any one of claims 1-9 wherein Z₂ issubstituted or unsubstituted morpholino, pyran, tetrahydropyran,piperazinyl, piperidinyl, tetrahydropyridinyl, pyrrolidinyl, pyrrolinyl,1,4-diazepanyl, or azepinyl.
 17. The process of any one of claims 1-16wherein the protein is selected from Bcl-2, Bcl-X_(L), and Mcl-1. 18.The process of any one of claims 1-17 wherein the protein is Bcl-X_(L).19. The process of any one of claims 1-17 wherein the protein is Mcl-1.20. An acylsulfonamide (3) having the formula:

wherein Z₁ has the formula:

Z₂ has the formula:

Z₁₁ and Z₁₃ are alkyl, substituted alkyl, —OH, —OR_(Z), —COOH,—COOR_(Z), —CONH₂, —NH₂, —NHR_(Z), —NR_(Z)R_(Z), —NO₂, —SH, —SR_(Z),—SO₂R_(Z), —SO₂H, —SOR_(Z), heterocyclo, and halo, among others, whereineach occurrence of R_(Z) is substituted or unsubstituted alkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedaralkyl; Z₂₂ is —N(Z₂₂₀)(Z₂₂₁) or —CH₂—N(Z₂₂₀)(Z₂₂₁), wherein Z₂₂₀ andZ₂₂₁ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, or Z₂₂₀ and Z₂₂₁ together with the nitrogen atom towhich they are attached, form a substituted or unsubstituted alicyclic,bicyclic, aryl, or heterocyclic moiety; and Z₁₀, Z₁₂, Z₁₄, Z₂₀, Z₂₁,Z₂₃, and Z₂₄ are hydrogen.